Matrices with memories

ABSTRACT

Combinations, called matrices with memories, of matrix materials that are encoded with an optically readable code are provided. The matrix materials are those that are used in as supports in solid phase chemical and biochemical syntheses, immunoassays and hybridization reactions. The matrix materials may additionally include fluophors or other luminescent moieties to produce luminescing matrices with memories. The memories include electronic and optical storage media and also include optical memories, such as bar codes and other machine-readable codes. By virtue of this combination, molecules and biological particles, such as phage and viral particles and cells, that are in proximity or in physical contact with the matrix combination can be labeled by programming the memory with identifying information and can be identified by retrieving the stored information. Combinations of matrix materials, memories, and linked molecules and biological materials are also provided. The combinations have a multiplicity of applications, including combinatorial chemistry, isolation and purification of target macromolecules, capture and detection of macromolecules for analytical purposes, selective removal of contaminants, enzymatic catalysis, cell sorting, sensors and drug delivery, chemical modification and other uses. Methods for tagging molecules, biological particles and matrix support materials, immunoassays, receptor binding assays, scintillation proximity assays, non-radioactive proximity assays, and other methods are also provided. Sensors containing a memory in combination with a matrix are also provided.

RELATED APPLICATIONS

This application is the National Stage of International PCT/US96/15999which designates the U.S. and was filed on Oct. 3, 1996 and published asWO97/12680, which is a continuation-in-part of U.S. application Ser. No.08/723,423, filed on Sep. 30, 1996, now issued as U.S. Pat. No.5,961,923, which is a continuation-in-part of U.S. application Ser. No.08/709,435, filed on Sep. 6, 1996, now issued as U.S. Pat. No.6,017,496, which is a continuation-in-part of U.S. application Ser. No.08/711,426, filed on Sep. 5, 1996, now issued as U.S. Pat. No.6,284,459, which is a continuation-in-part of U.S. application Ser. No.08/669,252, filed on Jun. 24, 1996, which is a continuation-in-part ofU.S. application Ser. No. 08/633,410, filed on Jun. 10, 1996, now issuedas U.S. Pat. No. 6,100,026, which is a continuation-in-part ofInternational PCT application No. PCT/US96/06145 which designates theU.S. and which was filed on Apr. 25, 1996 and published as WO 96/36436,and U.S. application Ser. No. 08/639,813, filed Apr. 2, 1996, nowabandoned, which is a continuation-in-part of U.S. application Ser. No.08/567,746, filed Dec. 5, 1995, now issued as U.S. Pat. No. 6,025,129,which is a continuation-in-part of U.S. application Ser. No. 08/538,387,filed Oct. 3, 1995, now issued as U.S. Pat. No. 5,874,214, which is acontinuation-in-part of each of U.S. application Ser. Nos. 08/480,147,08/484,486, 08/484,504, now issued as U.S. Pat. No. 5,751,629, Ser. No.08/480,196, now issued as U.S. Pat. No. 5,925,562, and Ser. No.08/473,660, all filed on Jun. 7, 1995, which are allcontinuations-in-part of U.S. application Ser. No. 08/428,662, filedApr. 25, 1995, now issued as U.S. Pat. No. 5,741,462.

The subject matter of each of above-noted U.S. applications andInternational PCT applications is incorporated herein by reference inits entirety.

FIELD OF THE INVENTION

The present invention relates to the application of information and datastorage and retrieval technology to molecular tracking andidentification and to biological, chemical, immunological andbiochemical assays.

BACKGROUND OF THE INVENTION

Automated identification of articles using bar codes in the availabilityof the integrated circuit technology and computing power at reasonablecosts. Such codes are typically used to track and identify consumergoods and other articles of manufacture. One of the first scannerscapable of reading a bar code was installed at a supermarket in 1974,and by 1980 more than 90% of all grocery items carried a bar code by1980. By December 1985, more than 12,000 grocery stores were equippedwith scanner checkout systems [See, e.g., Harmon et al. (1989) ReadingBetween the Lines-An Introduction to Bar Code Technology, HelmersPublishing, Inc. 1989]. Bar codes have also been used in otherapplications, including other inventory control systems and foridentification and characterization of responses to mass advertisingefforts.

By electro-optically scanning the symbol on an item and generating acorresponding signal, it is possible in an associated computer whosememory has digitally stored therein the full range of items, to comparethe signal derived from the scanned symbol with the stored information.When a match is found, the identity of the item and associatedinformation, such as, in the instance of consumer goods, its price. Thuscomputer technology is exploited to facilitate identification proceduresusing machine-readable identifiers.

Bar codes are typically read using lasers that scan from left to right,right to left, or in both directions (or other directions) across afield of alternating dark bars and reflective spaces of varying widths.Multiple scans are typically employed to minimize data errors. Becauseof the multiplicity of bars and spaces required for each alphanumericcharacter, bar codes generally require a relatively large space toconvey a small amount of data. For instance, each character in the barcode system known as Code 39 requires five bars and four spaces. A highdensity Code 39 field corresponds to only 9.4 characters per inch.Universal Product Codes (UPCs) are another common bar code usedprimarily in the retail grocery trade and contain a relatively largenumber of bars and spaces which allow for error checking, paritychecking and reduction of errors caused by manual scanning of articlesin grocery stores. They accordingly require even larger space forconveyance of character information. The Codabar code, which has beendeveloped by Pitney Bowes and is used in retail price labeling systemsand by Federal Express, is a self-checking code. Each character isrepresented by a stand-alone group of four bars and three interleavingspaces. Federal Express uses an eleven digit Codabar symbol on eachairbill to process more than 450,000 packages per night. Other codes usevarying bar and space techniques to represent characters. Because oferror checking requirements and for other reasons, however, the spacerequired to place a bar code on an article is relatively large.

In addition to the large surface area required for the series of barsand spaces that form a typical bar code symbol, the code must be placedon a background that has a high reflectance level. The high level ofcontrast, or reflectivity ratio, between the dark bars and thereflective spaces, allows the optical sensor in the reader to discernclearly and dependably the transitions between the bars and spaces inthe symbol. Ideally, the printed bar should be observed as perfectlyblack and the spaces should be perfectly reflective. Because those idealconditions are seldom possible, the industry typically requires thatlabeling media reflect at least 70% of incident light energy. Surfacereflectivity and thus quality of the media on which the bar code isplaced directly affects the successful use of the bar code on thatmedia. Additionally, the media cannot be overly transparent ortranslucent, since those characteristics can attenuate reflected light.Accordingly, only limited types of highly reflective media may be usedfor placement of bar codes. Space requirements for bar codes furtherinclude a “quiet zone” that surrounds the field of bars and spaces. Inmany codes, this quiet zone constitutes a border around the code symbol,thus requiring even more space for the bar code.

Bar coding also requires very precise print methods. Assuming that theprinting operation is capable of printing the required density toachieve the 70% reflectance ratio, careful attention must be paid toadditional major factors that influence the bar code effectiveness.These factors include ink spread/shrinkage; ink voids/specks, inksmearing; non-uniformity of ink; bar/space width tolerances; edgeroughness and similar factors that must be closely controlled to ensurethat the symbol will be easily scannable. In other words, the printermust pay careful attention to using paper or other media that displaysthe correct absorption properties properly inking the ribbon; carefullycontrolling hammer pressure; keeping the printhead and paper clean;properly wetting the paper and curing the ink; and maintaining properadjustment of the printhead control mechanism. These printing detailscreate additional problems and expenses, particularly for placement ofbar code symbols on smaller items such as coupons and mail pieces.

“Bar codes” containing an array of marks of any desired size and shapethat are arranged in a reference context or frame of one or more columnsand one or more rows, together with a reference marker and a referencecue have also been developed [see, U.S. Pat. No. 5,128,528]. The numberof rows corresponds to the number of characters contained in thesymbology selected for the array. For example, an array that is capableof conveying all the letters of the English language and ten numeralsymbols could use 36 rows. The number of columns in the matrix couldcorresponds to the number of characters desired to be conveyed. Theroles of the rows and columns in the reference frame may be reversed ifdesired. In the preferred embodiment, each column contains one or moredots corresponding to the character which is desired to be conveyed inthat column. The reference marker and reference cue may be formed of oneshape, of two marks, or according to any other desired arrangement thatallows interpretation of the matrix at any desired attitude with respectto the imaging equipment. The reference cue may form a part of thereference marker, or an information dot, if desired.

Thus, there are numerous types of bar codes, codes and methodologies foruse available. Bar coding and other coding technology, however, remainsto be fully exploited in areas outside the consumer products domain.Furthermore, other types of optical memories have not been exploited inany industry.

Drug Discovery

Drug discovery relies on the ability to identify compounds that interactwith a selected target, such as cells, an antibody, receptor, enzyme,transcription factor or the like. Traditional drug discovery relied oncollections or “libraries” obtained from proprietary databases ofcompounds accumulated over many years, natural products, fermentationbroths, and rational drug design. Recent advances in molecular biology,chemistry and automation have resulted in the development of rapid, Highthroughput screening (HTS) protocols to screen these collection. Inconnection with HTS, methods for generating molecular diversity and fordetecting, identifying and quantifying biological or chemical materialhave been developed. These advances have been facilitated by fundamentaldevelopments in chemistry, including the development of highly sensitiveanalytical methods, solid state chemical synthesis, and sensitive andspecific biological assay systems.

Analyses of biological interactions and chemical reactions, however,require the use of labels or tags to track and identify the results ofsuch analyses. Typically biological reactions, such as binding,catalytic, hybridization and signaling reactions, are monitored bylabels, such as radioactive, fluorescent, photoabsorptive, luminescentand other such labels, or by direct or indirect enzyme labels. Chemicalreactions are also monitored by direct or indirect means, such as bylinking the reactions to a second reaction in which a colored,fluorescent, chemiluminescent or other such product results. Theseanalytical methods, however, are often time consuming, tedious and, whenpracticed in vivo, invasive. In addition, each reaction is typicallymeasured individually, in a separate assay. There is, thus, a need todevelop alternative and convenient methods for tracking and identifyinganalytes in biological interactions and the reactants and products ofchemical reactions.

Combinatorial libraries

The provision and maintenance of compounds to support HTS have becomecritical. New and mthods for the lead generation and lead optimizationhave emerged to address this need for diversity. Among these methods iscombinatorial chemistry, which has become a powerful tool in drugdiscovery and materials science. Methods and strategies for generatingdiverse libraries, primarily peptide- and nucleotide-based oligomerlibraries, have been developed using molecular biology methods and/orsimultaneous chemical synthesis methodologies [see, e.g., Dower et al.(1991) Annu. Rep. Med. Chem. 26:271-280; Fodor et al. (1991) Science251:767-773; Jung et al. (1992) Angew. Chem. Ind. Ed. Engl. 31:367-383;Zuckerman et al. (1992) Proc. Natl. Acad. Sci. USA 89:4505-4509; Scottet al. (1990) Science 249:386-390; Devlin et al. (1990) Science249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA87:6378-6382; and Gallop et al. (1994) J. Medicinal Chemistry37:1233-1251]. The resulting combinatorial libraries potentially containmillions of pharmaceutically relevant compounds and that can be screenedto identify compounds that exhibit a selected activity.

The libraries fall into roughly three categories:fusion-protein-displayed peptide libraries in which random peptides orproteins are presented on the surface of phage particles or proteinsexpressed from plasmids; support-bound synthetic chemical libraries inwhich individual compounds or mixtures of compounds are presented oninsoluble matrices, such as resin beads [see, e.g., Lam et al. (1991)Nature 354:82-84] and cotton supports [see, e.g., Eichler et al. (1993)Biochemistry 32:11035-11041]; and methods in which the compounds areused in solution [see, e.g., Houghten et al. (1991) Nature 354:84-86,Houghten et al. (1992) BioTechniques 313:412-421; and Scott et al.(1994) Curr. Opin. Biotechnol. 5:40-48]. There are numerous examples ofsynthetic peptide and oligonucleotide combinatorial libraries. Thepresent direction in this area is to produce combinatorial librariesthat contain non-peptidic small organic molecules. Such libraries arebased on either a basis set of monomers that can be combined to formmixtures of diverse organic molecules or that can be combined to form alibrary based upon a selected pharmacophore monomer.

There are three critical aspects in any combinatorial library: (i) thechemical units of which the library is composed; (ii) generation andcategorization of the library, and (iii) identification of librarymembers that interact with the target of interest, and trackingintermediary synthesis products and the multitude of molecules in asingle vessel. The generation of such libraries often relies on the useof solid phase synthesis methods, as well as solution phase methods, toproduce collections containing tens of millions of compounds that can bescreened in diagnostically or pharmacologically relevant in vitro assaysystems. In generating large numbers of diverse molecules by stepwisesynthesis, the resulting library is a complex mixture in which aparticular compound is present at very low concentrations, so that it isdifficult or impossible to determine its chemical structure. Variousmethods exist for ordered synthesis by sequential addition of particularmoieties, or by identifying molecules based on spacial positioning on achip. These methods are cumbersome and ultimately impossible to apply tohighly diverse and large libraries. Identification of library membersthat interact with a target of interest, and tracking intermediarysynthesis products and the multitude of molecules in a single vessel isalso a problem. While considerable efforts have been devoted to thedevelopment of solid support chemistry, the choice of methods forstructural elucidation has been limited to spatial addressing, mixturedeconvolution, direct microanalysis and chemical tagging [see, e.g.,Metzger et al. (1994) Jung, Anal. Biochem. 219:261; Brown et al. (1995)Mol. Diversity 1:4; Youngquist et al. (1995) J. Am. Chem. Soc. 177:3900;Brummel et al. (1194) Science 264:399: Brenner et al. (1992) Proc. Natl.Acad. Sci. U.S.A. 89: 5381; Needles et al. (1993) Proc. Natl. Acad. Sci.U.S.A. 90:10700; Ohimeyer et al. Proc. Natl. Acad. Sci. U.S.A. 90:10922; Eckes (1994) Angew. Chem. Int. Ed. Engl. 33:1573; Ni et al.(1996) J. Med. Chem. 39:1601]. Tagging, especially non-chemical,non-invasive tagging, is potentially the most efficient and reliablestructural tracking method.

High Throughput Screening

In addition, exploitation of this diversity requires development ofmethods for rapidly screening compounds. Advances in instrumentation,molecular biology and protein chemistry and the adaptation ofbiochemical activity screens into microplate formats, has made itpossible to screen of large numbers-of compounds. Also, because compoundscreening has been successful in areas of significance for thepharmaceutical industry, high throughput screening (HTS) protocols haveassumed importance. Presently, there are hundreds of HTS systemsoperating throughout the world, which are used, not only for compoundscreening for drug discovery, but also for immunoassays, cell-basedassays and receptor-binding assays.

An essential element of high throughput screening for drug discoveryprocess and areas in which molecules are identified and tracked, is theability to extract the information made available during synthesis andscreening of a library, identification of the active components ofintermediary structures, and the reactants and products of assays. Whilethere are several techniques for identification of intermediary productsand final products, nanosequencing protocols that provide exactstructures are only applicable on mass to naturally occurring linearoligomers such as peptides and amino acids. Mass spectrographic [MS]analysis is sufficiently sensitive to determine the exact mass andfragmentation patterns of individual synthesis steps, but complexanalytical mass spectrographic strategies are not readily automated norconveniently performed. Also, mass spectrographic analysis provides atbest simple connectivity information, but no stereoisomeric information,and generally cannot discriminate among isomeric monomers. Anotherproblem with mass spectrographic analysis is that it requires purecompounds; structural determinations on complex mixtures is eitherdifficult or impossible. Finally, mass spectrographic analysis istedious and time consuming. Thus, although there are a multitude ofsolutions to the generation of libraries and to screening protocols,there are no ideal solutions to the problems of identification, trackingand categorization.

These problems arise in any screening or analytical process in whichlarge numbers of molecules or biological entities are screened. In anysystem, once a desired molecule(s) has been isolated, it must beidentified. Simple means for identification do not exist. Because of theproblems inherent in any labeling procedure, it would be desirable tohave alternative means for tracking and quantitating chemical andbiological reactions during synthesis and/or screening processes, andfor automating such tracking and quantitating.

Therefore, it is an object herein to provide methods for identification,tracking and categorization of the components of complex mixtures ofdiverse molecules. It is also an object herein to provide products forsuch identification, tracking and categorization and to provide assays,diagnostics and screening protocols that use such products. It is ofparticular interest herein to provide means to track and identifycompounds and to perform HTS protocols.

SUMMARY OF THE INVENTION

Combinations of matrix materials with programmable data storage orrecording devices or other memory means, herein referred to as memories,and assays using these combinations are provided. These combinations arereferred to herein as matrices with memories. Protocols in which allsteps, including synthesis and screening or assaying, are performed on asingle platform are provided herein. In addition protocols in which aseries of matrices with memories are used and information is transferredfrom one memory to another are also provided.

The memories with matrices may also include a sensing function thatrecords information related to the sample and/or source of the sampleand/or to detect or sense and store information about events inproximity the memory. In particular, sensors with memories are provided.

By virtue of the memory with matrix combination, molecules, such asantigens, antibodies, ligands, proteins and nucleic acids, andbiological particles, such as phage and viral particles and cells, thatare associated with, such as in proximity to or in physical contact withthe matrix combination or linked via information stored in a remotecomputer, can be electromagnetically tagged by programming the memorywith data corresponding to identifying information or can be tagged byimprinting or encoding the matrix with identifying information. Inparticular, optical memory means and the use thereof are providedherein.

Also provided herein are sensors that combine memories with matrices andsensors. Programming and reading the memory is effected remotely,preferably electromagnetic radiation, particularly radio frequency [RF]or radar, microwave, or microwave or energies between RF and microwave,or by reading the imprinted information.

Optical memories, either bar coded information, particularly the 2-D barcodes provided herein, or optically encoded memories, such as memoriesthat rely on changes in chemical or physical properties of particularmolecules are contemplated herein. Memories may also be remote from thematrix, such as instances in which the memory device is precoded with amark or identifier or the matrix is encoded with a bar code. Theidentity [i.e., the mark or code] of each device is written to a memory,which may be a computer or a piece of paper or any recording device, andinformation associated with each matrix is stored in the remote memoryand linked to or associated with the code or other identifier.

Of particular interest herein are matrices with memories in which thematrices have an engraved code. These matrices with memories are hereinreferred to as matrices with codes or optical memory devices [OMDs]. Thememories are remote recording devices, such as a remote computer memoryin which information associated with the codes is stored. The materialsare encoded with identifying information and/or any other information ofinterest. Synthetic protocols and assays using encoded matrix materialsare provided. By virtue of this code on the matrix, molecules, such asantigens, antibodies, ligands, proteins and nucleic acids, andbiological particles, such as phage and viral particles and cells, thatare associated with, such as in proximity to or in physical contact withthe matrix, can be tagged by programming a memory, such as a memory in acomputer, with data corresponding to the encoded identifyinginformation. The identity [i.e., the mark or code] of each device iswritten to a memory, which may be a computer or a piece of paper or anyrecording device, and information associated with each matrix is storedin the remote memory and linked to the code or other identifier.

The molecules and biological particles that are associated with thematrix combination, such as in proximity to or in physical contact orwith the matrix combination, can be identified and the results of theassays determined by retrieving the stored data points from thememories. Querying the memory will identify associated molecules orbiological particles that have reacted.

In certain embodiments of the matrices with memories, reactions, assaysand other events or external parameters, such as temperature and/or pH,can be monitored because occurrence of a reaction or an event can bedetected and such detection sent to the recording device when proximateto the matrix and recorded in the memory.

The combinations provided herein thus have a multiplicity ofapplications, including combinatorial chemistry, isolation andpurification of target macromolecules, capture and detection ofmacromolecules for analytical purposes, high throughput screening,selective removal of contaminants, enzymatic catalysis, drug delivery,chemical modification, information collection and management and otheruses. These combinations are particularly advantageous for use inmultianalyte analyses, assays in which a electromagnetic signal isgenerated by the reactants or products in the assay, for use inhomogeneous assays, and for use in multiplexed protocols.

In preferred embodiments, these matrix with memory combinations contain(i) a miniature recording device that includes one or more programmabledata storage devices [memories] or an engraved or imprinted opticallyreadable code or a 3-D optical memory, that can be remotely read and inpreferred embodiments also remotely programmed; and (ii) a matrix, suchas a particulate support used in chemical syntheses. These matrix withmemory combinations or the memories also are combined with sensors.

The matrix materials [matrices] are any materials that are routinelyused in chemical and biochemical synthesis. The matrix materials aretypically polymeric materials that are compatible with chemical andbiological syntheses and assays, and include, glasses, silicates,celluloses, polystyrenes, polysaccharides, polypropylenes, sand, andsynthetic resins and polymers, including acrylamides, particularlycross-linked polymers, cotton, and other such materials. The matricesmay be in the form of particles or may be continuous in design, such asa test tube or microplate, 96 well or 384 well or higher density formatsor other such microplates and microtiter plates. The matrices maycontain one or a plurality of recording devices. For example, each wellor selected wells in the microplate include a memory device in contacttherewith or embedded therein. The plates may further contain embeddedscintillant or a coating of scintillant [such as FlashPlate™, availablefrom DuPont NEN®, and plates available from Packard, Meriden, Conn. andCytostar-T plates available from Amersham International plc, U.K.].Automated robotic protocols will incorporate such plates for automatedmultiplexing [performing a series of coupled synthetic and processingsteps, typically, though not necessarily on the same platform, i.e.coupling of the chemistry to the biology] including one or more of thefollowing, synthesis, preferably accompanied by writing to the linkedmemories to identify linked compounds, screening, including usingprotocols with matrices with memories, and compound identification byquerying the memories of matrices associated with the selectedcompounds.

The matrices are either particulate of a size that is roughly about 1 to20 mm³ [or 1-20 mm in its largest dimension], preferably about 10 mm³ orsmaller, preferably 1 mm³ or smaller, such as minute particulates, or acontinuous medium, such as a microtiter plate, or other multi-wellplate, or plastic or other solid polymeric vial or glass vial orcatheter-tube [for drug delivery] or such container or deviceconventionally used in chemistry and biological syntheses and reactions.

In instances in which the matrix is continuous, the data storage device[memory] may be placed in, on, or under the matrix medium or may beembedded in the material of the matrix or removably attached, such as ina sleeve designed to fit on the matrix.

Plates that include a bar code, particularly the two-dimensional opticalbar code provided herein on the base of each well or elsewhere. Thetwo-dimensional bar code or other such code is particularly suited forapplication to each well in a microplate, such as a microtiter plate,that contain 96, 384, 1536 or higher density formats. The bar code mayalso be used in combination with modules that are fitted into the framesof 96 wells, or higher density formats [such as those available fromNUNC, such as NUNC-Immuno Modules, and also sources, such as COSTARplate strips, Cytostar-T plates from Amersham International plc, U.K.,and Octavac Filter Strips]. Separate containers or strips of containersare designed to fit into microplate frames. Each such container may beencoded with a bar code so that, upon removal from the strip, thecontainer, and thereby, its contents or history, may be identified.

In embodiments herein in which the matrices with memories are used inassays, such as scintillation proximity assays [SPA], FP [fluorescencepolarization] assays, FET [fluorescent energy transfer] assays, FRET[fluorescent resonance energy transfer] assays and HTRF [homogeneoustime-resolved fluorescence] assays, the matrices may be coated with,embedded with or otherwise combined with or in contact with assaymaterial, such as scintillant, fluophore or other fluorescent label. Theresulting combinations are called luminescing memories with matrices.When used in SPA formats they are referred to as scintillating matriceswith memories and when used in non-radioactive energy transfer formats[such as HTRF] they are referred to as fluorescing memories withmatrices.

The recording device used in proximity to the matrix is preferably aminiature device or is part of the support, such as an optical memory,typically less than 10-20 mm³ [or 10-20 mm in its largest dimension] insize, preferably smaller, such as 1 to 5 mm, that includes at least onedata storage unit that includes a remotely programmable and remotelyreadable, preferably non-volatile, memory. This device with remotelyprogrammable memory is in proximity to, associated with or in contactwith the matrix. In particular, the recording device includes a memorydevice, preferably having memory means, preferably non-volatile, forstoring a plurality of data points and means for receiving a transmittedsignal that is received by the device and for causing a data pointcorresponding to the data signal to be permanently stored within thememory means. If needed, the recording device further includes a shell[coating] that is non-reactive with and impervious to any processingsteps or solutions in which the combination of matrix with recordingdevice [matrix with memory] is placed, and that is transmissive of reador write signals transmitted to the memory. The device may also includeat least one support matrix disposed on an outer surface of the shellfor retaining molecules or biological particles. The shell and supportmatrix may be the same. In such instances, the shell must be treated orderivatized such that molecules, particularly amino acids and nucleicacids, can be linked, preferably either electrostatically or covalently,thereto. Thus, a transponder enclosed in plastic, must be furthertreated or coated to render it suitable for linkage of the molecule orbiological particle. In preferred embodiments provided herein, thedevice also includes a photodetector to detect events and means to writea record of the event to the memory.

The data storage device or memory is programmed with or encoded withinformation that identifies molecules or biological particles, either bytheir process of preparation, their identity, their batch number,category, physical or chemical properties, combinations of any of suchinformation, or other such identifying information. The molecules orbiological particles are in physical contact, direct or indirect, or inproximity with the matrix, which in turn is in physical contact or inthe proximity of the recording device that contains the data storagememory. The molecule or biological particle may also be associated, suchthat a molecule or biological particle that had been linked to or inproximity with a matrix with memory may be identified [i.e., althoughthe matrix particle and biological particle or molecule are not linkedor in proximity, the identify of the matrix that had been linked to themolecule or particle is known]. Typically, the matrix is on the surfaceof the recording device and the molecules and biological particles arein physical contact with the matrix material. In certain embodiments,the memory device may be linked to or in proximity to more than onematrix particle.

The data storage device or memory can also be programmed by virtue of areaction in proximity to or in the vicinity of the matrix with memory.In particular, the recording devices include memories and alsoadditional components that detect occurrence of external events or tomonitor the status of external parameters, such as EM emissions, changesin temperature or pH, ion concentrations and other such solutionparameters. For example, recording devices include memories and alsoinclude a photodetector can detect the occurrence of fluorescence orother optical emission. Coupling this emission with an amplifier andproviding a voltage to permit data storage in the matrix with memoryduring the reaction by way of, for example an RF signal transmitted toand received by an antenna/rectifier combination within the data storagedevice or providing voltage sufficient to write to memory from a battery[see, e.g., U.S. Pat. No. 5,350,645 and U.S. Pat. No. 5,089,877],permits occurrence of the emission to be recorded in the memory.

The recording device [containing the memory] is associated with thememory. Typically, the recording device is coated with at least onelayer of material, such as a protective polymer or a glass, includingpolystyrene, heavy metal-free glass, plastic, ceramic, and may be coatedwith more than one layers of this and other materials. It must betreated to render it suitable for linking molecules or biologicalparticles when it is used as a support. For example, it may be coatedwith a ceramic or glass that are suitably deivatized or then coated withor linked to the matrix material. Alternatively, the glass or ceramic orother coating may serve as the matrix. In other embodiments therecording device and the matrix material are in proximity, such as in acontainer of a size approximately that of the device and matrixmaterial. In yet other embodiments the recording device and matrixmaterial are associated, such that the molecule or biological particlethat was linked to the matrix or that was in proximity thereto may beidentified.

The matrix combinations [the matrices with memories], thus, contain amatrix material, typically in particulate form, in physical contact witha tiny device containing one or more remotely programmable data storageunits [memories]. Contact can be effected by placing the recordingdevice with memory on or in the matrix material or in a solution that isin contact with the matrix material or by linking the device, either bydirect or indirect covalent or non-covalent interactions, chemicallinkages or by other interactions, to the matrix. Alternatively,matrices with memories carry a code, such as a bar code, preferably atwo-dimensional bar code, on typically one surface and the memory isremote, such as a memory in a computer or any written record by whichthe code can be deciphered and information stored and associatedtherewith.

For example, when the memories are proximate to the matrix, contact canbe effected chemically, by chemically coupling the recording device withmemory to the matrix, or physically by coating the recording device withthe matrix material or another material, by physically inserting orencasing the device in the matrix material, by placing the device ontothe matrix or by any other means by which the device can be placed incontact with or in proximity to the matrix material. The contact may bedirect or indirect via linkers. The contact may be effected byabsorption or adsorption.

Since matrix materials have many known uses in conjunction withmolecules and biological particles, there are a multitude of methodsknown to artisans of skill in this art for linking, joining orphysically contacting the molecule or biological particle with thematrix material. In some embodiments, the recording device with datastorage unit is placed in a solution or suspension of the molecule orbiological particle of interest. In some of such instances, thecontainer, such as the microtiter plate or test tube or other vial, isthe matrix material. The recording device is placed in or on the matrixor is embedded, encased or dipped in the matrix material or otherwiseplace in proximity by enclosing the device and matrix material in asealed pouch or bag or container [MICROKAN™] fabricated from,preferably, porous material, such as polytetrafluoroethylene [marketedunder the trademark TEFLON® (Trademark, E. I. DuPont)] or polypropyleneprepared with pores, that is inert to the reaction of interest and thathave pores of size permeable to desired components of the reactionmedium.

More than one data storage device or engraved coded or combinationthereof may be in proximity to or contact with a matrix particle, ormore than one matrix particle may be in contact with on device. Forexample, microplates, such as microtiter y plates or other such highdensity format [i.e. 96, 384 1536 or more wells per plate, such as thoseavailable from Nunc, Naperville, Ill., Costar, Cambridge Mass. andMillipore, Bedford, Mass.] with the recording device containing the datastorage unit [remotely programmable memory] embedded in each well orvials [typically with a 1.5 ml or smaller capacity] with an embeddedrecording device may be manufactured.

In a preferred embodiment, the recording device is a semiconductor thatis approximately 10 mm or less in its largest dimension and the matrixmaterial is a particle, such as a polystyrene bead. The device and aplurality of particles, referred to as “beads”, typically about 1 mg toabout 50 mg, but larger size vessels and amounts up to 1000 mg,preferably 50 to about 200 mg, are sealed in chemically inert poroussupports, such as polypropylene formed so that it has pores of aselected size that excludes the particles but permits passage of theexternal medium. For example, a single device and a plurality ofparticles may be sealed in a porous or semi-permeable inert material toproduce a microvessel [such as the MICROKAN™] such as a TEFLON®[polytetrafluoroethylene] or polypropylene or membrane that is permeableto the components of the medium, or they may be contained in a smallclosable container that has at least one dimension that is porous or isa semi-permeable tube. Typically such tube, which preferably has an endthat can be opened and sealed or closed tightly.

These microvessels preferably have a volume of about 200-500 mm³, butcan have larger volumes, such as greater than 500 mm³ [or 1000 mm³] atleast sufficient to contain at least 200 mg of matrix particles, such asabout 500-3000 mm³, such as 1000-2000 or 1000 to 1500, with preferreddimensions of about 1-10 mm in diameter and 5 to 20 mm in height, morepreferably about 5 mm by 15 mm, or larger, such as about 1-6 cm by 1-6cm. The porous wall should be non-collapsible with a pore size in therange of 70 μM to about 100 μM, but can be selected to be semi-permeablefor selected components of the medium in which the microvessel isplaced. The preferred geometry of these combinations is cylindrical.These porous microvessels may be sealed by heat or may be designed tosnap or otherwise close. In some embodiments they are designed to bereused. In other embodiments, the microvessel MICROKAN™ with closuresmay be made out of non-porous material, such as a tube in the conicalshape or other geometry.

Such vessels thus are relatively rigid containers with mesh side walls.Typically, a single compound is synthesized in each one, and each onecontains a unique memory with encoded information or a read/write memoryand are designed to be loaded with solid phase resin. Syntheses takesplace by allowing reagents to flow through the outer mesh walls. Thepreferred embodiment has a volume of about 330 μl of which approximately200 μl is available for resin with the remainder of the space beingoccupied by the RF tag. In other embodiments the microvessel is engravedwith the 2-D optical code provided herien. Typically about 30 mg of mostcommercial resins can be loaded into this volume leaving enough spaceavailable for the resin to swell and still remain loose within thewalls.

Also provided herein are tubular devices (or other geometry) in whichthe recording device is enclosed in a solid polymer, such as apolypropylene, which is then radiation grafted with selected monomers toproduce a surface suitable for chemical synthesis and linkage ofmolecules or biological particles. These tubular devices (or othergeometry), such as the MICROTUBE™ microvessels, may contain a recordingdevice or may include a code engraved, such as by a laser, or otherwiseimprinted on the surface. The tubular devices are preferably TEFLON®(polytetrafluoroethylene (PTFE)), polyethylene, high densitypolyethylene, polypropylene, polystyrene, polyester, ceramic, compositesof any of these materials and other such materials. A method forradiation grafting of monomers to PTFE is provided herein. These devicesmay also be formed from a ball with a screw cap (MICROBALLS™) or othertype of cap to permit access to the inside.

These types of memories with matrices are polypropylene or fluoropolymertubes with a radiation grafted functionalized polystyrene surface thatcompletely enclose a selected memory, such as an RF tag. Syntheses areperformed on the functionalized polystyrene surface. These devices canbe used for solid phase chemistry without the need to load solid phaseresins.

Other devices of interest, are polypropylene supports, generally about5-10 mm in the largest dimension, and preferably a cube or other suchshape, that are marked with a code, and tracked using a remote memory.These microvessels can be marked with a code, such as a bar code,alphanumeric code, the 2-D optical bar code provided herein, or othermark or include an optical memory, for identification, particularly inembodiments in which the memory is not in proximity to the matrix, butis remote therefrom and used to store information regarding each codedvessel.

The combination of matrix with memory is used by contacting it with,linking it to, or placing it in proximity with a molecule or biologicalparticle, such as a virus or phage particle, a bacterium or a cell, toproduce a second combination of a matrix with memory and a molecule orbiological particle. In certain instances, such combinations of matrixwith memory or combination of matrix with memory and molecule orbiological particle may be prepared when used or may be prepared beforeuse and packaged or stored as such for futures use. The matrix withmemory when linked or proximate to a molecule or biological particle isherein referred to as a microreactor.

The miniature recording device containing the data storage unit(s) withremotely programmable memory, includes, in addition to the remotelyprogrammable memory, means for receiving information for storage in thememory and for retrieving information stored in the memory. Such meansis typically an antenna, which also serves to provide power in a passivedevice when combined with a rectifier circuit to convert receivedenergy, such as RF, into voltage, that can be tuned to a desiredelectromagnetic frequency to program the memory. Power for operation ofthe recording device may also be provided by a battery attached directlyto the recording device, to create an active device, or by other powersources, including light and chemical reactions, including biologicalreactions, that generate energy.

Preferred frequencies are any that do not substantially alter themolecular and biological interactions of interest, such as those thatare not substantially absorbed by the molecules or biological particleslinked to the matrix or in proximity of the matrix, and that do notalter the support properties of the matrix. Radio frequencies arepresently preferred, but other frequencies, such as radar, or opticallasers will be used, as long as the selected frequency or optical laserdoes not interfere with the interactions of the molecules or biologicalparticles of interest. Thus, information in the form of data pointscorresponding to such information is stored in and retrieved from thedata storage device by application of a selected electromagneticradiation frequency, which preferably is selected to avoid interferencefrom any background electromagnetic radiation.

The preferred miniature recording device for use in the combinationsherein is a single substrate of a size preferably less than about 10 to20 mm³ [or 10-20 mm in its largest dimension, most preferably 2 mm orless], that includes a remotely programmable data storage unit(s)[memory], preferably a non-volatile memory, and an antenna for receivingor transmitting an electromagnetic signal [and in some embodiments forsupplying power in passive devices when combined with a rectifiercircuit] preferably a radio frequency signal; the antenna, rectifiercircuit, memory and other components are preferably integrated onto asingle substrate, thereby minimizing the size of the device. An activedevice, i.e., one that does not rely on external sources for providingvoltage for operation of the memory, may include a battery for power,with the battery attached to the substrate, preferably on the surface ofthe substrate. Vias through the substrate can then provide conductionpaths from the battery to the circuitry on the substrate. The device israpidly or substantially instantaneously programmable, preferably inless than 5 seconds, more preferably in about 1 second, and morepreferably in about 50 to 100 milliseconds or less, and most preferablyin about 1 millisecond or less. In a passive device that relies uponexternal transmissions to generate sufficient voltage to operate, writeto and read from an electronic recording device, the preferred memory isnon-volatile, and may be permanent. Such memories may relyantifuse-based architecture or flash memory. Other memories, such aselectrically programmable erasable read only memories [EEPROMs] basedupon other architectures also can be used in passive devices. In activerecording devices that have batteries to assure continuous poweravailability, a broader range of memory devices may be used in additionto those identified above. These memory devices include dynamic randomaccess memories [DRAMS, which refer to semiconductor volatile memorydevices that allow random input/output of stored information; see, e.g.,U.S. Pat. Nos. 5,453,633, 5,451,896, 5,442,584, 5,442,212 and5,440,511], that permit higher density memories, and EEPROMs. Monolithicdevices, such as that described herein are among the preferredelectromagnetically programmable memories.

Containers, such as vials, tubes, microtiter plates, reagent bottles,sample and collection vials, autosampler carousals, HPLC columns andother chromatography columns, such as GC columns, electrophoresis andcapillary electrophoresis equipment, plate readers, reagent carriers,blood bags, fraction collectors, capsules and the like, which are incontact with a recording device that includes a data storage unit withprogrammable memory or include an optical memory, such as a 3-D opticalmemory incorporated into the material or attached to the container orinstrument, or other analytical tool are also provided. The memories mayalso be used in combination with instruments, including, but not limitedto HPLC, gas chromatographs (GC), mass spectrometers (MS), NMRinstruments, GC-MS, stir bars spectrometers, including fluorimeters,luminometers, and capillary electrophoresis and electrophoresisinstruments and tubes and plates used therefor. Thus, an entirelaboratory may be augmented with memories linked to or proximate toevery container, instrument, and device, from reagent bottle tocollected fraction, used in a particular protocol, whereby a sample maybe tracked. The information that is stored will include informationregarding the identity of a sample and/or source of the sample.

A container is typically of a size used in immunoassays or hybridizationreactions, generally a liter or less, typically less than 100 ml, andoften less than about 10 ml in volume, typically 100 μl-500 μl,particularly 200-250 μl. Alternatively the container can be in the formof a plurality of wells, such as a microtiter plate, each well havingabout 1 to 1.5 ml or less in volume. The container is transmissive tothe electromagnetic radiation, such as radio frequencies, infraredwavelengths, radar, ultraviolet wavelengths, microwave frequencies,visible wavelengths, X-rays or laser light, used to program therecording device.

The memories have also been combined with stirring bars, particularlymagnetic stir bars, thereby permitting remote identification of anybeakers, bottles, and other containers in which stir bars are used.

Methods for electromagnetically tagging molecules or biologicalparticles are provided. Such tagging is effected by placing themolecules or biological particles of interest in proximity with therecording device or with the matrix with memory, and programming orencoding the identity of the molecule or synthetic history of themolecules or batch number or other identifying information into thememory. The, thus identified molecule or biological particle is thenused in the reaction or assay of interest and tracked by virtue of itslinkage to the matrix with memory, its proximity to the matrix withmemory or its having been linked or in proximity to the matrix [i.e.,its association with], which can be queried at will to identify themolecule or biological particle. The tagging and/or reaction or assayprotocols may be automated. Automation may use robotics [see, U.S. Pat.No. 5,463,564, which provides an automated iterative method of drugdesign]. In addition, methods for addressing such memories inindividually among a group are provided.

In particular, methods for tagging constituent members of combinatoriallibraries and other libraries or mixtures of diverse molecules andbiological particles are provided. These methods involveelectromagnetically tagging or optically imprinting molecules,particularly constituent members of a library, by contacting themolecules or biological particles or bringing such molecules orparticles into proximity with a matrix with memory and programming thememory [by writing to it or by imprinting the matrix with an optical barcode or by associating a pre-engraved code with identifying informationwith retrievable information from which the identity, synthesis history,batch number or other identifying information can be retrieved. Thecontact is preferably effected by coating, completely or in part, therecording device with memory with the matrix and then linking, directlyor via linkers, the molecule or biological particle of interest to thematrix support. The memories can be coated with a protective coating,such as a glass or silicon, which can be readily derivatized forchemical linkage or coupling to the matrix material. In otherembodiments, the memories can be coated with matrix, such as for exampledipping the memory into the polymer prior to polymerization, andallowing the polymer to polymerize on the surface of the memory.

In other embodiments, the memory is part of the container that containsthe sample or is part of the instrument. As a sample is moved, forexample, from container to container or from instrument to container toa plate, the information from one memory is transferred by reading onememory and writing to the next so the identity of the contents istracked as it is processed. Such movement and tracking can be automated.

If the matrices are used for the synthesis of the constituent molecules,the memory of each particle is addressed and the identity of the addedcomponent is encoded in the memory at [before, during, or preferablyafter] each step in the synthesis. At the end of the synthesis, thememory contains a retrievable record of all of the constituents of theresulting molecule, which can then be used, either linked to thesupport, or following cleavage from the support in an assay or forscreening or other such application. If the molecule is cleaved from thesupport with memory, the memory must remain in proximity to the moleculeor must in some manner be traceable [i.e., associated with] to themolecule. Such synthetic steps may be automated.

In preferred embodiments, the matrix with memory with linked molecules[or biological particles] are mixed and reacted with a sample accordingto a screening or assay protocol, and those that react are isolated. Theidentity of reacted molecules can then be ascertained by remotelyretrieving the information stored in the memory and decoding it toidentify the linked molecules. Such steps can be performed on a singleplatform or on a series of platforms in which with each transferinformation from one memory is transferred to a subsequent memory thatis in contact with the sample.

Compositions containing combinations of matrices with memories andcompositions of matrices with memories and molecules or biologicalparticles are also provided. In particular, optically coded orelectronically tagged libraries of oligonucleotides, peptides, proteins,non-peptide organic molecules, phage display, viruses and cells areprovided. Particulate matrices, such as polystyrene beads, with attachedmemories, and continuous matrices, such as microtiter plates or slabs orpolymer, with a plurality of embedded or attached memories are provided.

These combinations of matrix materials with memories and combinations ofmatrices with memories and molecules or biological particles may be usedin any application in which support-bound molecules or biologicalparticles are used. Such applications include, but are not limited todiagnostics, such as immunoassays, drug screening assays, combinatorialchemistry protocols and other such uses. These matrices with memoriescan be used to tag cells for uses in cell sorting, to identify moleculesin combinatorial syntheses, to label monoclonal antibodies, to tagconstituent members of phage displays, affinity separation procedures,to label DNA and RNA, in nucleic acid amplification reactions [see,e.g., U.S. Pat. No. 5,403,484; U.S. Pat. No. 5,386,024; U.S. Pat. No.4,683,202 and, for example international PCT Application WO/94 02634,which describes the use of solid supports in connection with nucleicacid amplification methods], to label known compounds, particularlymixtures of known compounds in multianalyte analyses], to therebyidentify unknown compounds, or to label or track unknowns and therebyidentify the unknown by virtue of reaction with a known. Thus, thematrices with memories are particularly suited for high throughputscreening applications and for multianalyte analyses.

Systems and methods for recording and reading or retrieving theinformation in the data storage devices regarding the identity orsynthesis of the molecules or biological particles are also provided.The systems for recording and reading data include: a host computer orother encoder/decoder instrument having a memory for storing datarelating to the identity or synthesis of the molecules, and atransmitter means for receiving a data signal and generating a signalfor transmitting a data signal; and a recording device that includes aremotely programmable, preferably non-volatile, memory and transmittermeans for receiving a data signal and generating at least a transmittedsignal and for providing a write signal to the memory in the recordingdevice. The host computer stores transmitted signals from the memorieswith matrices, and decodes the transmitted information.

In particular, the systems include means for writing to and reading fromthe memory device to store and identify each of the indicators thatidentify or track the molecules and biological particles. The systemsadditionally include the matrix material in physical contact with orproximate to the recording device, and may also include a device forseparating matrix particles with memory so that each particle or memorycan be separately programmed.

Methods for tagging molecules and biological particles by contacting,either directly or indirectly, a molecule or biological particle with arecording device; transmitting from a host computer or decoder/encoderinstrument to the device electromagnetic radiation representative of adata signal corresponding to an indicator that either specifies one of aseries of synthetic steps or the identity or other information foridentification of the molecule or biological particle, whereby the datapoint representing the indicator is written into the memory, areprovided. Where optical memories are used the memories are opticallyscanned and the encoded information read.

Methods for reading identifying information from recording deviceslinked to or in contact with or in proximity to or that had been incontact with or proximity to a electromagnetically tagged molecule orelectromagnetically tagged biological particles are provided. Thesemethods include the step of exposing the recording device containing thememory in which the data are stored to electromagnetic radiation [EM];and transmitting to a host computer or decoder/encoder instrument anindicator representative of a the identity of a molecule or biologicalparticle or identification of the molecule or biological particle linkedto, in proximity to or associated with the recording device.

One, two, three and N-dimensional arrays of the matrices with memoriesare also provided. Each memory includes a record [or for pre-encodedmemories with matrices, the record is associated with code in a remotememory] of its position in the array. Such arrays may be used forblotting, if each matrix particle is coated on one at least one sidewith a suitable material, such as nitrocellulose. For blotting, eachmemory is coated on at least one side with the matrix material andarranged contiguously to adjacent memories to form a substantiallycontinuous sheet. After blotting, the matrix particles may be separatedand reacted with the analyte of interest [i., a labeled antibody oroligonucleotide or other ligand], after which the physical position ofthe matrices to which analyte binds may be determined. The amount ofbound analyte, as well as the kinetics of the binding reaction, may alsobe quantified. Southern, Northern, Western, dot blot and other suchassays using such arrays are provided. Dimensions beyond three can referto additional unique identifying parameters, such as batch number, andsimultaneous analysis of multiple blots.

Assays that use combinations of (i) a memory, such as a 2-D optical barcode or a miniature recording device that contains one or moreprogrammable data storage devices [memories] that can be remotelyprogrammed and read; and (ii) a matrix, such as a particulate supportused in chemical syntheses, are provided. The remote programming andreading is preferably effected using electromagnetic radiation.

Also provided are scintillation proximity assays, HTRF, FP, FET and FRETassays in which the memories are in proximity with or are in physicalcontact with the matrix that contains scintillant for detectingproximate radionucleotide signals or fluorescence. In addition,embodiments that include a memory device that also detects occurrence ofa reaction are provided.

Molecular libraries, DNA libraries, peptide libraries, biologicalparticle libraries, such as phage display libraries, in which theconstituent molecules or biological particles are combined with a solidsupport matrix that is combined with a data storage unit with aprogrammable memory are provided.

Affinity purification protocols in which the affinity resin is combinedwith a recording device containing a data storage unit with aprogrammable memory are also provided.

Immunological, biochemical, cell biological, molecular biological,microbiological, and chemical assays in which memory with matrixcombinations are used are provided. For example immunoassays, such asenzyme linked immunosorbent assays [ELISAs] in which at least oneanalyte is linked to a solid support matrix that is combined with arecording device containing a data storage unit with a programmable,preferably remotely programmable and non-volatile, memory are provided.

Of particular interest herein, are multiprotocol applications [such asmultiplexed assays or coupled synthetic and assay protocols] in whichthe matrices with memories are used in a series [more than one] ofreactions, a series [more than one] of assays, and/or a series of moreor more reactions and one or more assays, typically on a single platformor coupled via automated analysis instrumentation. As a result synthesisis coupled to screening, including compound identification and analysis,where needed.

As noted above, where sample is transferred, for example, from vial ortubes to plates, etc., the vials, plates, reagent bottles and columnsand other items used in drug discovery or for collecting and analyzingsamples, screening and analysis equipment and instrumentation includememory, such as an RF tag, optical memory, such as a 3-D optical memory,or 2-D optical bar code. As a sample is synthesized or obtained andprocessed, the information is transferred from one memory to the next,thereby providing a means to track the sample and identity fromsynthesis to screening to analysis.

Methods for engraving bar codes, bar codes and bar-code engraved devicesare also provided herein. In particular OMDs are provided and methodsfor writing to the surface of these devices and reading the engravedsymbology are provided. The OMDs are fabricated from a suitablematerial, such as black, white or colored glass, TEFLON®[polytetrafluoroethylene], polyethylene, high density polyethylene,polypropylene, polystyrene, polyester, ceramic, composites of any ofthese materials and other such materials. The typical OMD is 10 mm orsmaller in its largest dimension and is encoded by direct deposit, dotmatrix deposit, direct laser write or dot matrix scan laser write. Theymay be precoded or coded prior to or even during use. For use in theapplications provided herein, at least one surface or a portion of asurface is treated to render it suitable for use as a support, such asby grafting, ion implant, vacuum deposit, oxidation, combinationsthereof, suitable derivatization or any other means known to those ofskill in the art by which materials are treated to render them suitablefor use as supports. The OMDs also have applications as a data pad forrecording information about linked molecules or biological particles, orfor monitoring storage and location, or in clinical labs for recordingrelevant information. The OMDs may be in the form of microplates inwhich each well is encoded or in combination with any instrumentationused in biological and chemical processing and screening.

Also of particular interest herein, are combinations of vials, tubes orother containers with sleeves [see, e.g., FIGS. 35-38], in which vial[such as a Hewlett Packard or Waters HPLC vial] is inserted into afitted sleeve that contains the memory. These may be those used forpatient samples or HPLC samples or other samples, such as samples fromfraction collectors.

In one embodiment a carousel equipped with a reader and linked to acomputer with software is also provided. Also provided herein is thecarousel that houses a plurality of such vials or vessels, which areeach equipped with a memory device. The carousel is mounted on arotating seat that is designed to be rotated either manually, or byelectrical, mechanical or other suitable control. This seat is mountedto a housing and is positioned such that the carousel rotates with thememory device [i.e., a read/write device] coming in proximity to aread/write controller. This read/write controller is located within thehousing and positioned such that a detector head for the read/writecontroller is adjacent the read/write device as held in the carousel. Inorder to assist the accurate positioning of the carousel, a plunger isoriented on the surface of the housing to strike the carousel at thelocation of the vial which helps to prevent further rotation of thecarousel while the read/write device is communicating with theread/write controller. The read/write controller is a micro-controllerbased instrument that generates a selected frequency, such as 125 khzradio frequency (RF) signal, when RF devices are used, which istransmitted to the read/write controller head which includes an antennaelement that is designed to transmit the particular RF signal. It willbe appreciated that other electromagnetic frequencies, such asmicrowave, radar, x-ray, UV, and IR may be used.

Also included in the read/write controller is an oscillator and EEPROMmemory which, in combination, control a transmitter and receiver for therf signals. The read/write device includes a semiconductor that isattached to a similarly shaped antenna designed to receive the signalstransmitted from the read/write controller head. The signal from theread/write device antenna is filtered and a portion of the signal isrectified to create the power required to drive the semiconductor. Oncethe power is created, the semiconductor transmits through the sameantenna information, such as identifying information, that has beenprogrammed previously. This allows each vial to be attached to aread/write device, and programmed with a particular identification codeor other information.

In alternative embodiments, the read/write device is pre-programmed orthe container, such as the vial, reagent bottle, etc. is engraved withthe 2-D optical bar code provided herein and using the methods herein.The vial can be attached to the read/write device either before or afterthe vial is filled. Once the vial is attached to a read/write device,the vial can be inserted into the carousel with a number of other vialssimilarly attached to the read/write devices.

The information that is transmitted from the read/write device isreceived by the antenna in the read/write device head or is scanned withan optical reader. This received information is then analyzed by themicro-controller within the read/write device and the identificationcode is determined. This identification code is then output from theread/write controller via a serial data line which can be fed to acomputer. This output of the read/write controller is fed from theread/write controller to a computer system which identifies theparticular read/write device, and may combine the identificationinformation with the other information such as information regarding thecontents or source of the contents of the vial. Such information couldbe used to track the contents of the vial from location to locationwithin a lab, or to specifically identify a particular vial when thecontents of the vail are in question. Moreover, because of the virtuallyunlimited number of identification codes which could be programmed intothe read/write devices, an unlimited number of vials may be soidentified.

Devices specially adapted for opening and closing the microvessels, suchas the MICROKAN microvessels, are also provided.

Also provided herein are sensors in which matrices with memories areadapted to detect environmental parameters or changes or to be implantedin a mammal to detect internal parameters. Sensors containing memories,and coated with polymers and other materials that are responsive to theenvironment are also provided. In particular, sensors with memories thatare coated with electrically conducting polymers are provided.

Also provided are biosensors that are coated with angiogenic factors,such as vascular endothelial growth factors, basic and acid fibroblastgrowth factors, epidermal growth factors and interleukins are alsoprovided. Such angiogenic coating prevent encapsulation of such deviceswhen implanted in an animal.

Sol-gels containing memories and/or photodetectors and/or photodetectorsand memories are also provided. In particular, sol gels containingmemories and further adapted for use as sensors are also provided.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts combinatorial synthesis of chemical libraries on matrixsupports with memories. A, B, C . . . represent the chemical buildingblocks; a, b, c . . . represent the codes stored in memory thatcorrespond to each of A, B, C, . . . , respectively. S_(a), S_(b), S_(c). . . represent respective signals sent to memory. Alternatively, thematrix supports are OMDs [optical memory devices] that are encoded withsymbology associated with information stored in a remote memory, such asa computer. The symbology may be precoded or encoded prior to or duringsynthesis.

FIG. 2 depicts combinatorial synthesis of peptides on a matrix withmemory. Each amino acid has a corresponding code, a, b, c . . . , in thematrix memory, and L represents a Linker between the memory device andthe pharmacophore. Again as in FIG. 1, the matrix supports may beengraved with a code or symbology associated with information stored ina remote memory.

FIG. 3 depicts combinatorial synthesis of oligonucleotides on matrixsupports with memories. A, G, T and C represent nucleotides, and a, g,t, and c represent the electronic codes stored in memory that correspondto each of A, G T and C, respectively. The phosphoramidite method ofoligonucleotide synthesis is performed by methods known to those ofskill in the art [see, e.g., Brown et al. (1991) “Modern machine-aidedmethods of oligodeoxyribonucleotide synthesis” in OligonucleotidesAnalogues EDITOR: Eckstein, Fritz (Ed), IRL, Oxford, UK., pp. 1-24, esp.pp. 4-7]. As in FIGS. 1 and 2, the matrix may alternatively, oradditionally, have symbology engraved thereon.

FIG. 4 depicts generation of a chemical library, such as a library oforganic molecules, in which R₁, R₂, R₃ are substituents on selectedmolecule, such as a pharmacophore monomer, each identified with adifferent signal, depicted as 1, 2, or 3, from the classes S₁, S₂, S₃,respectively. The circle represents an organic pharmacophore. If R₁-R₃are the same, and selected from among the same 50 choices, then thecomplete library contains 50³=125,000 members. If R₁-R₃ selected fromamong different sets of choices, then the resulting library hascorrespondingly more members. Each optical memory device can be encodedwith information that represents the R_(n) added and class [S_(n)]thereby providing a unique code for each library member. As in FIGS.1-3, the matrix may be engraved with symbology, such as atwo-dimensional bar code.

FIG. 5 is a block diagram of the data storage means and supportingelectrical components of a preferred embodiment.

FIG. 6 is a diagrammatic view of the memory array within the recordingdevice, and the corresponding data stored in the host computer memory.

FIG. 7 is an illustration of an exemplary apparatus for separating thematrix particles with memories for individual exposure to an EM signal.

FIG. 8 is an illustration of a second exemplary embodiment of anapparatus for separating matrix particles for individual exposure to anoptical signal.

FIG. 9 is a diagrammatic view of the memory array within the recordingdevice, the corresponding data stored in the host computer memory, andincluded photodetector with amplifier and gating transistor.

FIG. 10 is a scheme for the synthesis of the 8 member RF encodedcombinatorial decameric peptide library described in EXAMPLE 4. Allcouplings were carried out in DMF at ambient temperature for 1 h [twocouplings per amino acid], using PyBOP and EDIA or DIEA. Deprotectionconditions: 20% piperidine in DMF, ambient temperature, 30 min; Cleavageconditions: 1,2-ethanedithiol:thioanisole:water:phenol:trifluoroaceticacid [1.5:3:3:4.5:88, w/w], ambient temperature, 1.5 h.

FIG. 11 is a side elevation of a preferred embodiment of a microvessel.

FIG. 12 is a sectional view, with portions cut away, taken along line12—12 of FIG. 11.

FIG. 13 is a sectional view taken along line 13—13 of FIG. 12.

FIG. 14 is a perspective view of an alternative embodiment of amicrovessel, with the end cap separated.

FIG. 15 is a side elevation view of the microvessel of FIG. 14, with aportion cut away.

FIG. 16 is a sectional view taken along line 16—16 of FIG. 15.

FIG. 17 is a perspective view of an exemplary write/read station.

FIG. 18 is a flow diagram of the operation of the system of FIG. 17.

FIG. 19 shows fluorescent solid supports: and their application in solidphase synthesis of direct SPA. The supports will include a either anelectromagnetically programmable memory, or an optical memory engravedon the surface, such as the 2-D optical bar code provided herein.

FIG. 20 Coded macro “beads” for efficient combinatorial synthesis. Aswith the supports, these “beads” will include either anelectromagnetically programmable memory, or an optical memory engravedon the surface, such as the 2-D optical bar code provided herein.

FIG. 21 Show the preparation and use of a tubular microvessel in whichthe container is radiation grafted with monomers or otherwise activatedfor use as a support matrix. As with the supports and “beads”, these“beads” will include either an electromagnetically programmable memory,or an optical memory on the surface, such as the 2-D optical bar codeprovided herein.

FIG. 22 is a perspective view of a first embodiment of an optical memorydevice;

FIG. 23 is an exploded perspective view of a second embodiment of theoptical memory device;

FIG. 24 is a diagrammatic view of the optical write and read for theoptical memory devices;

FIG. 25 is a side elevation of a third embodiment of the optical memorydevice;

FIG. 26 is a side elevation of a fourth embodiment of the optical memorydevice;

FIG. 27 is a side elevation of a fifth embodiment of the optical memorydevice;

FIG. 28 is a front elevation of a sixth embodiment of the optical memorydevice;

FIG. 29 is a front elevation of a seventh embodiment of the opticalmemory device;

FIG. 30 is a front elevation of an eighth embodiment of the opticalmemory device;

FIG. 31 is a flow diagram of the image processing sequence for atwo-dimensional bar code on an optical memory device; and

FIG. 32 is a diagrammatic view of an exemplary handling system forfeeding, reading and distributing the optical memory devices.

FIGS. 33A-E depict the OMDs with optical symbology provided herein. FIG.33A illustrates an exemplary OMD. FIG. 33B depicts a close-up of a 2-Dlaser etched bar code that is read by the software described herein thatreads the code in two dimensions, horizontally and verticallysimultaneously using a camera and pattern recognition software describedherein. With reference to the exemplified embodiment [see EXAMPLES], thecode in this figure is 0409AA55AA550409. The blacked out and whitenedsquares represent data units. Etching of the entire 2-D bar code by aCO₂ laser can be accomplished with a resolution below about 0.5 mm. FIG.33C depicts a split and pool combinatorial synthesis protocol using theOMDs and directed sorting. A, B and C represent building blocks, and thenumbers above each OMD represent a 2-D optical bar code [single digitsare used merely for exemplification]. FIG. 33D depicts synthesis of a3×3×3 oligonucleotide library using the OMDs and directed sorting[reaction conditions are described in the EXAMPLES; DMT-X₂, X₃ or X₄ is5′-O-DMT-2′deoxyadenosine-3′-O-phosphoramidite,5′-O-DMT-2′deoxycytidine-3′-O-phosphoramidite,5′-O-DMT-2′deoxyguanosine-3′-O-phosphoramidite; B₂, B₃ or B₄ is adenine,cytosine or guanine. FIG. 33E depicts an oligonucleotide hexamer libraryusing the optical memory device. Each B^(n) refers to a nucleoside base,and the resulting library, where n=6, will contain 4096 unique members[4⁶=4096].

FIGS. 34A-D depicts a protocol for radiation grafting of polymers to theinert surfaces to render them suitable for use as matrices. FIG. 34Aexemplifies the grafting of a polymer to a tube containing an RF tag,linkage of scintillant to the surface, organic synthesis and then use ofthe resulting compound linked to the support in an assay. Thus, allsteps are performed on the same platform. FIG. 34B also exemplifies asingle platform protocol. FIG. 34C depicts the preparation of a tubulardevices in which the matrix is the radiation grafted PTFE and the memoryis a transponder, such as the BMDS transponder or IDTAG™ transponder[such as a MICROTUBE], described herein; FIG. 34D depicts a small chip[2 mm×2 mm×0.1 mm] encased in a radiation grafted polypropylene orteflon ball [ball or bead or other such geometry] with a screw cap [suchas a MICROBALL or MICROBEAD].

FIG. 35 is a perspective view of an alternative embodiment of aexemplary read/write station;

FIG. 36 is a plan view of the read/write station shown in FIG. 35;

FIG. 37 is a cross-sectional view of the read/write station taken alongline 37—37 of FIG. 35;

FIG. 38 is a perspective view of a cylindrical tube having a read/writedevice attached to its lower end;

FIG. 39 is a cross-sectional view of the cylindrical tube and read/writedevice taken along line 39—39 of FIG. 38;

FIG. 40 is an enlarged cross-sectional view of the read/write deviceshowing the coil antenna, microcircuit, and housing;

FIG. 41 is a perspective view of the microcircuit shown attached to asubstrate;

FIG. 42 is a perspective view of the sealing apparatus which is used tocreate an environmental on the lower end of the housing;

FIG. 43 is a view of a typical program output to the screen showing thedecoded identification code, in combination with a graphicalrepresentation of the contents of the cylindrical tube.

FIG. 44 is a side elevation view of the capsule sealing tool mounted ona plier type tool;

FIG. 45 is an enlarged sectional view taken on line 47—47 of FIG. 46;

FIG. 46 is a sectional view taken along line 48—48 of FIG. 47, showingthe initial cap engagement;

FIG. 47 is a view similar to a portion of FIG. 46, showing the cap fullyseated;

FIG. 48 is a view similar to a portion view of FIG. 46, showing ejectionof the sealed capsule;

FIG. 49 is a side elevation view of the uncapping tool;

FIG. 50 is a sectional view taken on line 52—52 of FIG. 49, showing thecap separated from the capsule;

FIG. 51 is a perspective view of a monolithic identification tag withthe antenna formed on the substrate;

FIG. 52 is a plan view of the monolithic identification tag as shown inFIG. 51, showing generally the outline of the circuitry on thesubstrate, and the formation of the antenna on encircling thatcircuitry; and

FIG. 53 is a perspective view of a typical stirring bar with portions ofthe encapsulation removed to show the positioning of a monolithicidentification tag against the metal of the stirring bar.

FIG. 54 is a circuit diagram of the basic components and interconnectionof an exemplary sensor with memory;

FIG. 55 is a simple circuit diagram for a temperature sensor;

FIG. 56 is a block diagram of the components of an exemplary implantableglucose sensing system;

FIG. 57 is a cross-section of an exemplary implantable device withlogic, power and communication electronics with electrode sensors;

FIG. 58 is a perspective diagrammatic view of the sensor assembly of anexemplary implantable glucose sensing system;

FIG. 59 is a flow diagram the basic control software for the exemplaryimplantable glucose sensing system;

FIG. 60 is a block diagram of the components of an exemplaryintracranial pressure monitor;

FIG. 61 is a diagrammatic view of an exemplary optical intracranialpressure monitor;

FIG. 62 is a diagrammatic view of an alternate optical sensor for use inthe intracranial pressure monitor;

FIG. 63 is a diagrammatic view of an exemplary urea sensor located inline with a hemodialysis system;

FIG. 64 is a diagrammatic view of an exemplary embodiment of a “smart”blood bag;

FIG. 65 is a diagrammatic view of an alternate optical sensor for use ina “smart” blood bag; and

FIG. 66 is a diagrammatic view of electrode construction for analternate embodiment of a glucose sensor.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are, unless noted otherwise, incorporated byreference in their entirety. In the event a definition in this sectionis not consistent with definitions elsewhere, the defintion set forth inthis section will control.

As used herein, a bar codes refers any array optically readable marks ofany desired size and shape that are arranged in a reference context orframe of, preferably, although not necessarily, one or more columns andone or more rows. For purposes herein, the bar code refers to anysymbology, not necessary “bar” but may include dots, characters or anysymbol or symbols.

As used herein, an optical memory refers to the symbology and thesurface on which it is engraved or otherwise imprinted or refers toother optical devices. For purposes herein, an optical memory alsoincludes from optical recording media that may be appropriate for use inthe recording devices and combinations herein and include, but are notlimited to, optical discs, magneto-optical materials, photochromicmaterials, photoferroelectric materials, and photoconductiveelectro-optic materials. Optical memories also include memories, such as2-D and 3-D optical memories that use optics, such as lasers, forwriting and/or reading.

As used herein, an optical memory device [OMD] refers to a surface thatis encoded with a code, preferably the 2-D bar code provided herein. Foruse herein, such devices include at least two surfaces, one of which istreated or formed from a matrix material treated to render it suitablefor use as a support to which molecules or biological particles arelinked, such as in chemical syntheses or as supports in assays, and theother that includes a code that can be optically read and then comparedwith information in a computer or other memory to interpret its meaning.

As used herein, symbology refers to the code, such as a bar code, thatis engraved or imprinted on the OMD. The symbology is any code known ordesigned by the user. The symbols are associated with information storedin a remote computer or memory or other such device or means. Forexample, each OMD can be uniquely identified with an encoded symbology.The process steps or additions or manipulations to the associatedmolecules or biological particles can be recorded in a remote memory andassociated with the code.

As used herein, a matrix refers to any solid or semisolid or insolublesupport on which a code is to which the memory device and/or themolecule of interest, typically a biological molecule, organic moleculeor biospecific ligand is linked or contacted. Typically a matrix is asubstrate material having a rigid or semi-rigid surface. In manyembodiments, at least one surface of the substrate will be substantiallyflat, although in some embodiments it may be desirable to physicallyseparate synthesis regions for different polymers with, for example,wells, raised regions, etched trenches, or other such topology. Matrixmaterials include any materials that are used as affinity matrices orsupports for chemical and biological molecule syntheses and analyses,such as, but are not limited to: polystyrene, polycarbonate,polypropylene, nylon, glass, dextran, chitin, sand, pumice,polytetrafluoroethylene, agarose, polysaccharides, dendrimers,buckyballs, polyacrylamide, Kieselguhr-polyacrlamide non-covalentcomposite, polystyrene-polyacrylamide covalent composite,polystyrene-PEG [polyethyleneglycol] composite, silicon, rubber, andother materials used as supports for solid phase syntheses, affinityseparations and purifications, hybridization reactions, immunoassays andother such applications. The matrix herein may be particulate or may bein the form of a continuous surface, such as a microtiter dish or well,a glass slide, a silicon chip with a surface adapted for linking ofbiological particles or molecules, a nitrocellulose sheet, nylon mesh,or other such materials. When particulate, typically the particles haveat least one dimension in the 5-10 mm range or smaller. Such particles,referred collectively herein as “beads”, are often, but not necessarily,spherical. Such reference, however, does not constrain the geometry ofthe matrix, which may be any shape, including random shapes, needles,fibers, elongated, etc. The “beads” may include additional components,such as magnetic or paramagnetic particles [see, e.g., Dyna beads(Dynal, Oslo, Norway)] for separation using magnets, fluophores andother scintillants, as long as the additional components do notinterfere with chemical reactions, data entry or retrieval from thememory.

Significantly, it is noted, however, that many surfaces, such as glass,require modification to render them suitable for use as supports. Anysuch surface must be treated to render it suitable for chemicalsyntheses or for adsorption of biological particles. Chemical synthesesrequire a support that not only has the proper surface characteristics(organic solvent wettability, chemical kinetics, etc.), but that alsohas a high density of functional groups. An untreated glass surfacecontains only a very small amount [less than 1 nmol/sq. mm] of hydroxygroups. It is also very hydrophilic and not very suitable for reactionsin organic media. Therefore, the glass surface has to be modified toachieve high functional group density (˜>10 nmol/mm²) and properhydrophobicity. Thus, as used herein, matrix refers to materials thathave been so-treated. Therefore, a transponder in which the memorydevice is encased in a glass capsule for instance is not usable as is,but must be treated, either by coating at least one surface with apolymer, such as by grafting, derivatizing or otherwise activating thesurface.

As used herein, scintillants include, 2,5-diphenyloxazole [PPO],anthracene, 2-(4′-tert-butylphenyl)-5-(4″-biphenyl)-1,3,4-oxadiazole[butyl-PBD]; 1-phenyl-3-mesityl-2-pyrazoline [PMP], with or withoutfrequency shifters, such as 1,4,-bis[5-phenyl(oxazolyl)benzene] [POPOP];p-bis-o-methylstyrylbenzene [bis-MSB]. Combinations of these fluors,such as PPO and POPOP or PPO and bis-MSB, in suitable solvents, such asbenzyltoluene [see, e.g., U.S. Pat. No. 5,410,155], are referred to asscintillation cocktails.

As used herein a luminescent moiety refers to a scintiilant or fluophorused in scintillation proximity assays or in non-radioactive energytransfer assays, such as HTRF assays.

As used herein, fluorescent resonance energy transfer [FRET] is anart-recognized term meaning that one fluorophore [the acceptor] can bepromoted to an excited electronic state through quantum mechanicalcoupling with and receipt of energy from an electronically excitedsecond fluorophore [the donor]. This transfer of energy results in adecrease in visible fluorescence emission by the donor and an increasein fluorescent energy emission by the acceptor. Significant energytransfer can only occur when the donor and acceptor are sufficientlyclosely positioned since the efficiency of energy transfer is highlydependent upon the distance between donor and acceptor fluorophores.

As used herein, matrix particles refer to matrix materials that are inthe form of discrete particles. The particles have any shape anddimensions, but typically have at least one dimension that is 100 mm orless, preferably 50 mm or less, more preferably 10 mm or less, andtypically have a size that is 100 mm³ or less, preferably 50 mm³ orless, more preferably mm³ or less, and most preferably 1 mm³ or less.The matrices may also be continuous surfaces, such as microtiter plates[e.g., plates made from polystyrene or polycarbonate or derivativesthereof commercially available from Perkin Elmer Cetus and numerousother sources, and Covalink trays [Nunc], microtiter plate lids or atest tube, such as a 1 ml Eppendorf tube or smaller versions, such as500 μl, 200 μl or smaller. Matrices that are in the form of containersrefers to containers, such as test tubes and microplates and vials thatare typically used for solid phase syntheses of combinatorial librariesor as pouches, vessels, bags, and microvessels for screening anddiagnostic assays or as containers for samples, such as patient samples.Thus, a container used for chemical syntheses refers to a container thattypically has a volume of about 1 liter, generally 100 ml, and moreoften 10 ml or less, 5 ml or less, preferably 1 ml or less, and as smallas about 50 μl-500 μl, such as 100 μl or 250 μl or 200 μl. This alsorefers to multi-well plates, such as microtiter plates [96 well, 384well, 1536 well or other higher density format]. Such microplate willtypically contain a memory device in, on, or otherwise in contact within each of a plurality of wells.

As used herein, a matrix with a memory refers to a combination of amatrix with a miniature recording device that stores multiple bits ofdata by which the matrix may be identified, preferably in a non-volatilememory that can be written to and read from by transmission ofelectromagnetic radiation from a remote host, such as a computer. Byminiature is meant of a size less than about 10-20 mm³ [or 10-20 mm inthe largest dimension]. Preferred memory devices or data storage unitsare miniature and are preferably smaller than 10-20 mm³ [or 10-20 mm inits largest dimension] dimension, more preferably less than 5 mm³, mostpreferably about 1 mm³ or smaller. Alternatively, the memory may befabricated as part of the matrix material or may be a chemical orbiological-based memory means, such as those described herein, includingthe rhodopsin based memories and 3-D optical memories based onphotochromic materials [see, e.g., U.S. Pat. Nos. 5,268,862, 5,130,362,5,325,324; see, also, Dvornikov et al. (1996) Opt. Commun. 128:205-210;

Dvornikov et al. (1996) Res. Chem. Intermed. 22:115-28; Dvornikov et al.(1994) Proc. SPIE-Int. Soc. Opt. Eng. 2297:447-51; Dvornikov et al.(1994) Mol. Cryst. Lig. Cryst. Sci. Technol., Sect. A 246:379-88;Dvornikov et al. (1994) J. Phys. Chem. 98:6746-52; Ford et al. (1993)Proc. SPIE-Int. Soc. Opt. 2026:604-613; Ford et al. Proc. SPIE-Int. Soc.Opt. Eng. 1853:5-13; Malkin et al. Res. Chem. Intermed. 19:159-89;Dvornikov et al. (1993) Proc. SPIE-Int. Soc. Opt. Eng. 1852:243-52;Dvornikov et al. (1992) Proc. SPIE-Int. Soc. Opt. Eng. 1662:197-204;Prasad et al. (1996) Mater. Res. Soc. Symp. Proc. 413:203-213].

As used herein, a microreactor refers to combinations of matrices withmemories with associated, such as linked or proximate, biologicalparticles or molecules. It is produced, for example, when the moleculeis linked thereto or synthesized thereon. It is then used in subsequentprotocols, such as immunoassays and scintillation proximity assays.

As used herein, a combination herein called a microvessel [e.g., amicrovessel such as those designated presently designated a MICROKAN™]refers to a combination in which a single device for more than onedevice] and a plurality of particles are sealed in a porous orsemi-permeable inert material, such as polytetrafluoroethylene orpolypropylene or membrane that is permeable to the components of themedium, but retains the particles and memory, or are sealed in a smallclosable container that has at least one dimension that is porous orsemi-permeable. Typically such microvessels, which preferably have atleast one end that can be opened and sealed or closed tightly, has avolume of about 200-500 mm³, with preferred dimensions of about 1-10 mmin diameter and 5 to 20 mm in height, more preferably about 5 mm by 15mm. The porous wall should be non-collapsible with a pore size in therange of 70 μM to about 100 μM, but can be selected to be semi-permeablefor selected components of the reaction medium.

As used herein, a memory is a data storage unit [or medium] withprogrammable memory, preferably a non-volatile memory; or alternativelyis a symbology on a surface, such as a bar code, whose identity and asfor which associate information is stored in a remote memory, such as acomputer memory.

As used herein, programming refers to the process by which data orinformation is entered and stored in a memory. A memory that isprogrammed is a memory that contains retrievable information.

As used herein, remotely programmable, means that the memory can beprogrammed without direct physical or electrical contact or can beprogrammed from a distance, typically at least about 10 mm, althoughshorter distances may also be used, such as instances in which theinformation comes from surface or proximal reactions or from an adjacentmemory or in instances, such as embodiments in which the memories arevery close to each other, as in microtiter plate wells or in an array.

As used herein, a recording device [or memory device] is an apparatusthat includes the data storage unit with programmable memory, and, ifnecessary, means for receiving information and for transmittinginformation that has been recorded. It includes any means needed or usedfor writing to and reading from the memory. The recording devicesintended for use herein, are miniature devices that preferably aresmaller than 10-20 mm³ [or 10-20 mm in their largest dimension], andmore preferably are closer in size to 1 mm³ or smaller that contain atleast one such memory and means for receiving and transmitting data toand from the memory. The data storage device also includes opticalmemories, such as bar codes, on devices such as OMDs.

As used herein, a data storage unit with programmable memory includesany data storage means having the ability to record multiple discretebits of data, which discrete bits of data may be individually accessed[read] after one or more recording operations. Thus, a matrix withmemory is a combination of a matrix material with a data storage unit.

As used herein, programmable means capable of storing unique datapoints. Addressable means having unique locations that may be selectedfor storing the unique data points.

As used herein, reaction verifying and reaction detecting areinterchangeable and refer to the combination that also includes elementsthat detect occurrence of a reaction or event of interest between theassociated molecule or biological particle and its environment [i.e.,detects occurrence of a reaction, such as ligand binding, by virtue ofemission of EM upon reaction or a change in pH or temperature or otherparameter].

As used herein, a host computer or decoder/encoder instrument is aninstrument that has been programmed with or includes information [i.e.,a key] specifying the code used to encode the memory devices. Thisinstrument or one linked thereto transmits the information and signalsto the recording device and it, or another instrument, receives theinformation transmitted from the recording device upon receipt of theappropriate signal. This instrument thus creates the appropriate signalto transmit to the recording device and can interpret transmittedsignals. For example, if a “1” is stored at position 1,1 in the memoryof the recording device means, upon receipt of this information, thisinstrument or computer can determine that this means the linked moleculeis, for example, a peptide containing alanine at the N-terminus, anorganic group, organic molecule, oligonucleotide, or whatever thisinformation has been predetermined to mean. Alternatively, theinformation sent to and transmitted from the recording device can beencoded into the appropriate form by a person.

As used herein, an electromagnetic tag is a recording device that has amemory that contains unique data points that correspond to informationthat identifies molecules or biological particles linked to, directly orindirectly, in physical contact with or in proximity [or associatedwith] to the device. Thus, electromagnetic tagging is the process bywhich identifying or tracking information is transmitted [by any meansand to any recording device memory, including optical and magneticstorage media] to the recording device.

As used herein, proximity means within a very short distance, generallyless than 0.5 inch, typically less than 0.2 inches. In particular,stating that the matrix material and memory, or the biological particleor molecule and matrix with memory are in proximity means that, they areat least or at least were in the same reaction vessel or, if the memoryis removed from the reaction vessel, the identity of the vesselcontaining the molecules or biological particles with which the memorywas proximate or linked is tracked or otherwise known.

As used herein, associated with means that the memory must remain inproximity to the molecule or biological particle or must in some mannerbe traceable to the molecule or biological particle. For example, if amolecule is cleaved from the support with memory, the memory must insome manner be identified as having been linked to the cleaved molecule.Thus, a molecule or biological particle that had been linked to or inproximity to a matrix with memory is associated with the matrix ormemory if it can be identified by querying the memory.

As used herein, antifuse refers to an electrical device that isinitially an open circuit that becomes a closed circuit duringprogramming, thereby providing for non-volatile memory means and, whenaccompanied by appropriate transceiver and rectification circuitry,permitting remote programming and, hence identification. In practice, anantifuse is a substantially nonconductive structure that is capable ofbecoming substantially conductive upon application of a predeterminedvoltage, which exceeds a threshold voltage. An antifuse memory does notrequire a constant voltage source for refreshing the memory and,therefore, may be incorporated in a passive device. Other memories thatmay be used include, but are not limited to: EEPROMS, DRAMS and flashmemories.

As used herein, flash memory is memory that retains information whenpower is removed [see, e.g., U.S. Pat. No. 5,452,311, U.S. Pat. No.5,452,251 and U.S. Pat. No. 5,449,941]. Flash memory can be rewritten byelectrically and collectively erasing the stored data, and then byprogramming.

As used herein, passive device refers to an electrical device which doesnot have its own voltage source and relies upon a transmitted signal toprovide voltage for operation.

As used herein, electromagnetic [EM] radiation refers to radiationunderstood by skilled artisans to be EM radiation and includes, but isnot limited to radio frequency [RF], infrared [IR], visible, ultraviolet[UV], radiation, sonic waves, X-rays, and laser light.

As used herein, information identifying or tracking a biologicalparticle or molecule, refers to any information that identifies themolecule or biological particle, such as, but not limited to theidentity particle [i.e. its chemical formula or name], its sequence, itstype, its class, its purity, its properties, such as its bindingaffinity for a particular ligand. Tracking means the ability to follow amolecule or biological particle through synthesis and/or process steps.The memory devices herein store unique indicators that represent any ofthis information.

As used herein, combinatorial chemistry is a synthetic strategy thatproduces diverse, usually large, chemical libraries. It is thesystematic and repetitive, covalent connection of a set, the basis set,of different monomeric building blocks of varying structure to eachother to produce an array of diverse molecules [see, e.g., Gallop et al.(1994) J. Medicinal Chemistry 37:1233-1251]. It also encompasses otherchemical modifications, such as cyclizations, eliminations, cleavages,etc., that are carried in manner that generates permutations and therebycollections of diverse molecules.

As used herein, a biological particle refers to a virus, such as a viralvector or viral capsid with or without packaged nucleic acid, phage,including a phage vector or phage capsid, with or without encapsulatednucleotide acid, a single cell, including eukaryotic and prokaryoticcells or fragments thereof, a liposome or micellar agent or otherpackaging particle, and other such biological materials.

As used herein, the molecules in the combinations include any molecule,including nucleic acids, amino acids, other biopolymers, and otherorganic molecules, including peptidomimetics and monomers or polymers ofsmall organic molecular constituents of non-peptidic libraries, that maybe identified by the methods here and/or synthesized on matrices withmemories as described herein.

As used herein, the term “bio-oligomer” refers to a biopolymer of lessthan about 100 subunits. A bio-oligomer includes, but is not limited to,a peptide, i.e., containing amino acid subunits, an oligonucleotide,i.e., containing nucleoside subunits, a peptide-oligonucleotide chimera,peptidomimetic, and a polysaccharide.

As used herein, the term “sequences of random monomer subunits” refersto polymers or oligomers containing sequences of monomers in which anymonomer subunit may precede or follow any other monomer subunit.

As used herein, the term “library” refers to a collection ofsubstantially random compounds or biological particles expressing randompeptides or proteins or to a collection of diverse compounds. Ofparticular interest are bio-oligomers, biopolymers, or diverse organiccompounds or a set of compounds prepared from monomers based on aselected pharmacophore.

As used herein, an analyte is any substance that is analyzed or assayedin the reaction of interest. Thus, analytes include the substrates,products and intermediates in the reaction, as well as the enzymes andcofactors.

As used herein, multianalyte analysis is the ability to measure manyanalytes in a single specimen or to perform multiple tests from a singlespecimen. The methods and combinations herein provide means to identifyor track individual analytes from among a mixture of such analytes.

As used herein, a fluophore or a fluor is a molecule that readilyfluoresces; it is a molecule that emits light following interaction withradiation. The process of fluorescence refers to emission of a photon bya molecule in an excited singlet state. For scintillation assays,combinations of fluors are typically used. A primary fluor that emitslight following interaction with radiation and a secondary fluor thatshifts the wavelength emitted by the primary fluor to a higher moreefficiently detected wavelength.

As used herein, a peptidomimetic is a compound that mimics theconformation and certain stereochemical features of the biologicallyactive form of a particular peptide. In general, peptidomimetics aredesigned to mimic certain desirable properties of a compound but not theundesirable features, such as flexibility leading to a loss of thebiologically active conformation and bond breakdown. For example,methylenethio bioisostere [CH₂S] has been used as an amide replacementin enkephalin analogs [see, e.g., Spatola, A. F. Chemistry andBiochemistry of Amino Acids, Peptides, and Proteins [Weinstein, B, Ed.,Vol. 7, pp. 267-357, Marcel Dekker, New York (1983); and Szelke et al.(1983) In Peptides: Structure and Function, Proceedings of the EighthAmerican Peptide Symposium, Hruby and Rich, Eds., pp. 579-582, PierceChemical Co., Rockford, Ill.].

As used herein, complete coupling means that the coupling reaction isdriven substantially to completion despite or regardless of thedifferences in the coupling rates of individual components of thereaction, such as amino acids In addition, the amino acids, or whateveris being coupled, are coupled to substantially all available couplingsites on the solid phase support so that each solid phase support willcontain essentially only one species of peptide.

As used herein, the biological activity or bioactivity of a particularcompound includes any activity induced, potentiated or influenced by thecompound in vivo or in vitro. It also includes the abilities, such asthe ability of certain molecules to bind to particular receptors and toinduce [or modulate] a functional response. It may be assessed by invivo assays or by in vitro assays, such as those exemplified herein.

As used herein, pharmaceutically acceptable salts, esters or otherderivatives of the compounds include any salts, esters or derivativesthat may be readily prepared by those of skill in this art using knownmethods for such derivatization and that produce compounds that may beadministered to animals or humans without substantial toxic effects andthat either are pharmaceutically active or are prodrugs. For example,hydroxy groups can be esterified or etherified.

As used herein, substantially pure means sufficiently homogeneous toappear free of readily detectable impurities as determined by standardmethods of analysis, such as thin layer chromatography [TLC], massspectrometry [MS], size exclusion chromatography, gel electrophoresis,particularly agarose and polyacrylamide gel electrophoresis [PAGE] andhigh performance liquid chromatography [HPLC], used by those of skill inthe art to assess such purity, or sufficiently pure such that furtherpurification would not detectably alter the physical and chemicalproperties, such as enzymatic and biological activities, of thesubstance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound may, however, be amixture of stereoisomers. In such instances, further purification mightincrease the specific activity of the compound.

As used herein, adequately pure or “pure” per se means sufficiently purefor the intended use of the adequately pure compound.

As used herein, biological activity refers to the in vivo activities ofa compound or physiological responses that result upon in vivoadministration of a compound, composition or other mixture. Biologicalactivity, thus, encompasses therapeutic effects and pharmaceuticalactivity of such compounds, compositions and mixtures.

As used herein, a prodrug is a compound that, upon in vivoadministration, is metabolized or otherwise converted to thebiologically, pharmaceutically or therapeutically active form of thecompound. To produce a prodrug, the pharmaceutically active compound ismodified such that the active compound will be regenerated by metabolicprocesses. The prodrug may be designed to alter the metabolic stabilityor the transport characteristics of a drug, to mask side effects ortoxicity, to improve the flavor of a drug or to alter othercharacteristics or properties of a drug. By virtue of knowledge ofpharmacodynamic processes and drug metabolism in vivo, those of skill inthis art, once a pharmaceutically active compound is known, can designprodrugs of the compound [see, e.g., Nogrady (1985) Medicinal ChemistryA Biochemical Aproach, Oxford University Press, New York, pages388-392].

As used herein, amino acids refer to the naturally-occurring amino acidsand any other non-naturally occurring amino acids, and also thecorresponding D-isomers. It is also understood that certain amino acidsmay be replaced by substantially equivalent non-naturally occurringvariants thereof, such as D-Nva, D-Nle, D-Alle, and others listed withthe abbreviations below or known to those of skill in this art.

As used herein, hydrophobic amino acids include Ala, Val, Leu, lle, Pro,Phe, Trp, and Met, the non-naturally occurring amino acids and thecorresponding D isomers of the hydrophobic amino acids, that havesimilar hydrophobic properties; the polar amino acids include Gly, Ser,Thr, Cys, Tyr, Asn, Gln, the non-naturally occurring amino acids and thecorresponding D isomers of the polar amino acids, that have similarproperties, the charged amino acids include Asp, Glu, Lys, Arg, His, thenon-naturally occurring amino acids and the corresponding D isomers ofthese amino acids.

As used herein, Southern, Northern, Western and dot blot proceduresrefer to those in which DNA, RNA and protein patterns, respectively, aretransferred for example, from agarose gels, polyacrylamide gels or othersuitable medium that constricts convective motion of molecules, tonitrocellulose membranes or other suitable medium for hybridization orantibody or antigen binding are well known to those of skill in this art[see, e.g., Southern (1975) J. Mol. Biol. 98:503-517; Ketner et al.(1976) Proc. Natl. Acad. Sci. U.S.A. 73:1102-1106; Towbin et al. (1979)Proc. Natl. Acad. Sci. U.S.A. 76:4350].

As used herein, a receptor refers to a molecule that has an affinity fora given ligand. Receptors may be naturally-occurring or syntheticmolecules. Receptors may also be referred to in the art as anti-ligands.As used herein, the terms, receptor and anti-ligand are interchangeable.Receptors can be used in their unaltered state or as aggregates withother species. Receptors may be attached, covalently or noncovalently,or in physical contact with, to a binding member, either directly orindirectly via a specific binding substance or linker. Examples ofreceptors, include, but are not limited to: antibodies, cell membranereceptors surface receptors and internalizing receptors, monoclonalantibodies and antisera reactive with specific antigenic determinants[such as on viruses, cells, or other materials], drugs, polynucleotides,nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides,cells, cellular membranes, and organelles.

Examples of receptors and applications using such receptors, include butare not restricted to:

a) enzymes: specific transport proteins or enzymes essential to survivalof microorganisms, which could serve as targets for antibiotic [ligand]selection;

b) antibodies: identification of a ligand-binding site on the antibodymolecule that combines with the epitope of an antigen of interest may beinvestigated; determination of a sequence that mimics an antigenicepitope may lead to the development of vaccines of which the immunogenis based on one or more of such sequences or lead to the development ofrelated diagnostic agents or compounds useful in therapeutic treatmentssuch as for auto-immune diseases

c) nucleic acids: identification of ligand, such as protein or RNA,binding sites;

d) catalytic polypeptides: polymers, preferably polypeptides, that arecapable of promoting a chemical reaction involving the conversion of oneor more reactants to one or more products; such polypeptides generallyinclude a binding site specific for at least one reactant or reactionintermediate and an active functionality proximate to the binding site,in which the functionality is capable of chemically modifying the boundreactant [see, e.g., U.S. Pat. No. 5,215,899];

e) hormone receptors: determination of the ligands that bind with highaffinity to a receptor is useful in the development of hormonereplacement therapies; for example, identification of ligands that bindto such receptors may lead to the development of drugs to control bloodpressure; and

f) opiate receptors: determination of ligands that bind to the opiatereceptors in the brain is useful in the development of less-addictivereplacements for morphine and related drugs.

As used herein, antibody includes antibody fragments, such as Fabfragments, which are composed of a light chain and the variable regionof a heavy chain.

As used herein, complementary refers to the topological compatibility ormatching together of interacting surfaces of a ligand molecule and itsreceptor. Thus, the receptor and its ligand can be described ascomplementary, and furthermore, the contact surface characteristics arecomplementary to each other.

As used herein, a ligand-receptor pair or complex formed when twomacromolecules have combined through molecular recognition to form acomplex.

As used herein, an epitope refers to a portion of an antigen moleculethat is delineated by the area of interaction with the subclass ofreceptors known as antibodies.

As used herein, a ligand is a molecule that is specifically recognizedby a particular receptor. Examples of ligands, include, but are notlimited to, agonists and antagonists for cell membrane receptors, toxinsand venoms, viral epitopes, hormones [e.g., steroids], hormonereceptors, opiates, peptides, enzymes, enzyme substrates, cofactors,drugs, lectins, sugars, oligonucleotides, nucleic acids,oligosaccharides, proteins, and monoclonal antibodies.

As used herein, a sensor is a device or apparatus that monitors external(or internal) parameters (i.e., conditions), such as ion concentrations,pH, temperatures, and events. Internal parameters refer to conditions,concentrations, such as electrolyte and glucose concentration, in ananimal. Biosensors are sensors that detect biological species. Sensorsencompass devices that rely on electrochemical, optical, biological andother such means to monitor the environment.

As used herein, multiplexing refers to performing a series of syntheticand processing steps and/or assaying steps on the same platform [i.e.solid support or matrix] or coupled together as part of the sameautomated coupled protocol, including one or more of the following,synthesis, preferably accompanied by writing to the linked memories toidentify linked compounds, screening, including using protocols withmatrices with memories, and compound identification by querying thememories of matrices associated with the selected compounds. Thus, theplatform refers system in which all manipulations are performed. Ingeneral it means that several protocols are coupled and performedsequentially or simultaneously.

As used herein, a platform refers to the instrumentation or devices inwhich on which a reaction or series of reactions is(are) performed.

As used herein a protecting group refers to a material that ischemically bound to a monomer unit that may be removed upon selectiveexposure to an activator such as electromagnetic radiation and,especially ultraviolet and visible light, or that may be selectivelycleaved. Examples of protecting groups include, but are not limited to:those containing nitropiperonyl, pyrenylmethoxy-carbonyl, nitroveratryl,nitrobenzyl, dimethyl dimethoxybenzyl, 5-bromo-7-nitroindolinyl,o-hydroxy- alpha -methyl cinnamoyl, and 2-oxymethylene anthraquinone.

Also protected amino acids are readily available to those of skill inthis art. For example, Fmoc and Boc protected amino acids can beobtained from Fluka, Bachem, Advanced Chemtech, Sigma, CambridgeResearch Biochemical, Bachem, or Peninsula Labs or other chemicalcompanies familiar to those who practice this art.

As used herein, the abbreviations for amino acids and protective groupsare in accord with their common usage and the IUPAC-IUB Commission onBiochemical Nomenclature [see, (1972) Biochem. 11: 942-944]. Eachnaturally occurring L-amino acid is identified by the standard threeletter code or the standard three letter code with or without the prefix“L-”; the prefix “D-” indicates that the stereoisomeric form of theamino acid is D. For example, as used herein, Fmoc is9-fluorenylmethoxycarbonyl; BOP isbenzotriazol-1-yloxy-tris(dimethylamino) phosphoniumhexafluorophosphate, DCC is dicyclohexyl-carbodiimide; DDZ isdimethoxydimethylbenzyloxy; DMT is dimethoxytrityl; FMOC isfluorenylmethyloxycarbonyl; HBTU is2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium; hexafluorophosphateNV is nitroveratryl; NVOC is 6-nitro-veratryloxycarbonyl and otherphotoremovable groups; TFA is trifluoroacetic acid; DMF forN,N-dimethylformamide; Boc is tert-butoxycarbonyl; ACN is acetonitrile,TFA for trifluoroacetic acid; HF for hydrogen fluoride; HFIP forhexafluoroisopropanol; HPLC for high performance liquid chromatography;FAB-MS for fast atom bombardment mass spectrometry; DCM isdichloromethane, Bom is benzyloxymethyl; Pd/C is palladium catalyst onactivated charcoal; DIC is diisopropylcarbodiimide; DCC isN,N′-dicyclohexylcarbodiimide; [For] is formyl; PyBop isbenzotriazol-1-yl-oxy-trispyrrolidino-phosphonium hexafluoro-phosphate;POPOP is 1,4,-bis[5-phenyl(oxazolyl)benzenel; PPO is2,5-diphenyloxazole; butyl-PBD is[2-(4′-tert-butylphenyl)-5-(4″-biphenyl)-1,3,4-oxadiazole]; PMP is(1-phenyl-3-mesityl-2-pyrazoline) DIEA is diisopropylethyl-amine; EDIAis ethyidiisopropylethylamine; NMP is N-methylpyrrolidone; NV isnitroveratryl PAL is pyridylalanine; HATU isO(7-azabenzotriaol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate;TFA is trifluoracetic acid, THF is tetrahydrofuran; and EDT is1,2-ethanedithiol.

A. Matrices

For purposes herein matrices refer to supports used to retain moleculesand biological particles, such as for chemical synthesis and tocontainers, such as microplates and test tubes. Matrices used forsupports will be derivatized or otherwise suitable for retainingmolecules or biological particles. Containers will either be derivatizedor otherwise suitable for retaining molecules or biological particles orwill be suitable for containing molecules and biological particles. Inthe embodiments of interest herein, the matrices are engraved with anoptical bar code, include a memory and are associated with a sensor orare associated with containers, laboratory equipment and other suchdevices.

Matrices, which are generally insoluble materials used to immobilizeligands and other molecules, have application in many chemical synthesesand separations. Matrices are used in affinity chromatography, in theimmobilization of biologically active materials, and during chemicalsyntheses of biomolecules, including proteins, amino acids and otherorganic molecules and polymers. The preparation of and use of matricesis well known to those of skill in this art; there are many suchmaterials and preparations thereof known. For example,naturally-occurring matrix materials, such as agarose and cellulose, maybe isolated from their respective sources, and processed according toknown protocols, and synthetic materials may be prepared in accord withknown protocols.

Matrices include any material that can act as a support matrix forattachment of the molecules or biological particles of interest and canbe in contact with or proximity to or associated with, preferablyencasing or coating, the data storage device with programmable memory.Any matrix composed of material that is compatible with and upon or inwhich chemical syntheses are performed, including biocompatiblepolymers, is suitable for use herein. The matrix material should beselected so that it does not interfere with the chemistry or biologicalreaction of interest during the time which the molecule or particle islinked to, or in proximity therewith [see, e.g., U.S. Pat. No.4,006,403]. These matrices, thus include any material to which the datastorage device with memory can be attached, placed in proximity thereof,impregnated, encased or otherwise connected, linked or physicallycontacted. Such materials are known to those of skill in this art, andinclude those that are used as a support matrix. These materialsinclude, but are not limited to, inorganics, natural polymers, andsynthetic polymers, including, but are not limited to: cellulose,cellulose derivatives, acrylic resins, glass that is derivatized torender it suitable for use a support, silica gels, polystyrene, gelatin,polyvinyl pyrrolidone, co-polymers of vinyl and acrylamide, polystyrenecross-linked with divinylbenzene or the like [see, Merrifield (1964)Biochemistry 3:1385-1390], polyacrylamides, latex gels, polystyrene,dextran, polyacrylamides, rubber, silicon, plastics, nitrocellulose,celluloses, natural sponges, and many others. It is understood that thematrix materials contemplated are those that are suitable for use a ssupport matrix for retaining molecules or biological particles duringsyntheses or reactions.

Among the preferred matrices are polymeric beads, such as the TENTAGEL™resins and derivatives thereof [sold by Rapp Polymere, Tubingen,Germany; see, U.S. Pat. No. 4,908,405 and U.S. Pat. No. 5,292,814; see,also Butz et al. (1994) Peptide Res. 7:20-23; Kleine et al. (1994)Immunobiol. 190:53-66; see, also Piskin et al. (1994), Chapter 18“Nondegradable and Biodegradable Polymeric Particles” in DiagnosticBiosensor Polymers, ACS Symp. Series 556, Usmani et al. Eds, AmericanChemical Society, Washington, DC], which are designed for solid phasechemistry and for affinity separations and purifications. See, alsoBayer et al. (1994) in Pept.: Chem., Struct. Biol., Proc. Am. Pept.Symp., 13th; Hodges, et al. eds., pp.156-158; Zhang et al. (1993) Pept.1992, Proc. Eur. Pent. Symp., 22nd, Schneider, et al., eds. pp. 432-433;llg et al. (1994) Macromolecules, pp. 2778-83; Zeppezauer et al. (1993)Z. Naturforsch., B: Chem. Sci. 48:1801-1806; Rapp et al. (1992) Pept.Chem. 1992, Proc. Jpn. Symp., 2nd, Yanaihara, ed., pp. 7-10; Nokihara etal. (1993) Shimadzu Hyoron 50:25-31; Wright et al. (1993) TetrahedronLett. 34:3373-3376; Bayer et al. (1992) Poly(Ethylene Glycol) Chem.Harris, ed., pp. 325-45; Rapp et al. (1990) Innovation Perspect. SolidPhase Synth. Collect. Pap., Int. Symp., 1st, Epton, ed., pp. 205-10;Rapp et al. (1992) Pept.: Chem. Biol., Proc. Am. Pept. Symp., 12th,Smith et al., eds., pp. 529-530; Rapp et al. (1989) Pept., Proc. Eur.Pept. Symp., 20th, Jung et al., ed., pp. 199-201; Bayer et al. (1986)Chem. pept. Proteins 3: 3-8; Bayer et al. (1983) Pept.: Struct. Funct.,Proc. Am. pept. Symp., 8th, Hruby et al. eds., pp. 87-90 fordescriptions of preparation of such beads and use thereof in syntheticchemistry. Matrices that are also contemplated for use herein includefluophore-containing or -impregnated matrices, such as microplates andbeads [commercially available, for example, from Amersham, ArlingtonHeights, Ill; plastic scintillation beads from NE (Nuclear Technology,Inc., San Carlos, Calif.), Packard, Meriden, Conn.]. It is understoodthat these commercially available materials will be modified bycombining them with memories, such as by methods described herein.

The matrix may also be a relatively inert polymer, which can be graftedby ionizing radiation [see, e.g., FIG. 21, which depicts a particularembodiment] to permit attachment of a coating of polystyrene or othersuch polymer that can be derivatized and used as a support. Radiationgrafting of monomers allows a diversity of surface characteristics to begenerated on plasmid supports [see, e.g., Maeji et al. (1994) ReactivePolymers 22:203-212; and Berg et al. (1989) J. Am. Chem. Soc. 111:8024-8026]. For example, radiolytic grafting of monomers, such as vinylmonomers, or mixtures of monomers, to polymers, such as polyethylene andpolypropylene, produce composites that have a wide variety of surfacecharacteristics. These methods have been used to graft polymers toinsoluble supports for synthesis of peptides and other molecules, andare of particular interest herein. The recording devices, which areoften coated with a plastic or other insert material, can be treatedwith ionizing radiation so that selected monomers can be grafted torender the surface suitable for chemical syntheses.

Where the matrix particles are macroscopic in size, such as about atleast 1 mm in at least one dimension, such bead or matrix particle orcontinuous matrix may contain one or more memories. Where the matrixparticles are smaller, such as NE particles [PVT-based plasticscintillator microsphere], which are about 1 to 10 μm in diameter, morethan one such particle will generally be associated with one memory.Also, the “bead” or plate or container may include additional material,such as scintillant or a fluophore impregnated therein. In preferredembodiments, the solid phase chemistry and subsequent assaying may beperformed on the same bead or matrix with memory combination. Allprocedures, including synthesis on the bead and assaying and analysis,can be automated.

The matrices are typically insoluble substrates that are solid, porous,deformable, or hard, and have any required structure and geometry,including, but not limited to: beads, pellets, disks, capillaries,hollow fibers, needles, solid fibers, random shapes, thin films andmembranes. Typically, when the matrix is particulate, the particles areat least about 10-2000 μM, but may be smaller, particularly for use inembodiments in which more than one particle is in proximity to a memory.For purposes herein, the support material will typically encase or be incontact with the data storage device, and, thus, will desirably have atleast one dimension on the order of 1 mm [1000 μM] or more, althoughsmaller particles may be contacted with the data storage devices,particularly in embodiments in which more than one matrix particle isassociated, linked or in proximity to one memory or matrix with memory,such as the microvessels [see, e.g., FIGS. 11-16]. Each memory will bein associated with, in contact with or proximity to at least one matrixparticle, and may be in contact with more than one. As smallersemiconductor and electronic or optical devices become available, thecapacity of the memory can be increased and/or the size of the particlescan be decreased. For example, presently, 0.5 micron semiconductordevices are available. Integrated circuits 0.25-micron in size have beendescribed and are being developed using a technology called theComplementary Metal Oxide-Semiconductor process (see, e.g., Investor'sBusiness Daily May 30, 1995).

Also of interest herein, are devices that are prepared by inserting therecording device into a “tube” [see, e.g., FIG. 21] or encasing them inan inert material [with respect to the media in which the device will bein contact]. This material is fabricated from a plastic or other inertmaterial. Preferably prior to introducing [and preferably sealing] therecording device inside, the tube or encasing material is treated withionizing radiation to render the surface suitable for grafting selectedmonomers, such as styrene [see, e.g., Maeji et al. (1994) ReactivePolymers 22:203-212; Ang et al. in Chapter 10: Application of RadiationGrafting in Reagent Insolubilization, pp 223-247; and Berg et al. (1989)J. Am. Chem. Soc. 111:8024-8026].

Recording device(s) is(are) introduced inside the material or thematerial is wrapped around the device and the resulting matrix withmemory “tubes” [MICROTUBES™, see, FIG. 21] are used for chemicalsynthesis or linkage of selected molecules or biological particles.These “tubes” are preferably synthesized from an inert resin, such as apolypropylene resin [e.g., a Moplen resin, V29G PP resin from Montell,Newark Del., a distributor for Himont, Italy]. Any inert matrix that canthen be functionalized or to which derivatizable monomers can be graftedis suitable. Preferably herein, polypropylene tubes are grafted and thenformed into tubes or other suitable shape and the recording deviceinserted inside. These tubes [MICROTUBES™] with grafted monomers arethen used as synthesis, and/or for assays or for multiplexed processes,including synthesis and assays or other multistep procedures.

Such tubes may also have snap on or screw tops so that the memory deviceor chip is removable. For example, they may be conical tubes likeEppendorf tubes, with a snap on top, preferably a flat top. The tubeswill be of a size to accommodate a memory device and thus may be assmall as about 2 mm×2 mm×0.1 to hold the small 2 mm×2 mm×0.1 mm devicedescribed herein. They will be fabricated from polypropylene or othersuitable material and radiation grafted, see above, and Examples, below,preferably prior to introduction of the memory device.

Solid tubular embodiments, such as the MICROTUBE microreactors made oftubes for other geometry] have been coated or grafted with suitablematerials are used as a solid support for any other methods disclosedherein, including organic syntheses and assays. Fluorophores,scintillants and other such compounds may also be incorporated into thesurface or linked thereto [see, EXAMPLES below]. These tubes includethose that contain the memory encased either permanently or removably orthat include an imprinted symbology.

Briefly, for radiation-induced graft copolymerization, for example, ofstyrene to polypropylene (PP), polyethylene (PE) and teflon (PTFE)tubes, the diameter of the tube can be any desired size, with 0.1 mm to20 mm presently preferred and 2 mm to 5 mm more preferred. It has beenfound that dilution of styrene with methanol enhances the rate ofgrafting, thereby permitting use of PTFE tubes. Dilutions, which can bedetermined empirically for each material, from 5% to 70% have beentested. PTFE and PE tubes have the highest styrene grafting at a 50%dilution, and polypropylene tubes have the best performance when graftedat a 35% dilution. To effect grafting the polymer tubes are irradiatedunder a Co⁶⁰ source. The dose rate can be empirically determined. Ratesof 0.01×10⁶ to 1×10⁶ rads (r)/h are typical and the most effective ratewas 0.1×10⁶ r/h. A total dose of 0.5-10×10⁶ rads was typical and themost effective dose was 2.6-2.9×10⁶ rads.

Functional groups are introduced by selection of the monomers, such asstyrene, choloromethylstyrene, methylacrylate, 2-hydroxymethylacrylateand/or other vinyl monomers containing one or more functional groups.For example (see, e.g., FIG. 33) aminomethyl functional groups beenintroduced by first radiation grafting polystyrene onto the surface oftubes of the above-noted polymers tubes followed by functionalizationusing N-(hydroxymethyl)phthal-imide with trifluoromethanesulfonic acidas a catalyst. The polystyrene grafted polymer tube is thoroughly washedbefore use to remove residual monomer, non-attached polystyrene andadditives remaining from the radiation grafting. The amidoalkylationproceeds smoothly at room temperature in 50% (v/v) trifluoroaceticacid-dichloromethane solvent for 24 hours. Loading can be controlled bychanging the concentrations of reagent, catalyst and/or reaction time.Hydrazinolysis in refluxing ethanol gives the aminomethyl polystyrenegrafted polymer tube. Adjustable loading range is on the order of0.5-100 μmol per tube, depending the size of the tube and the polymer.

A carboxylic acid group was introduced by using acrylate acid orfunctionalization of polystyrene. The polystyrene grafted tube wasfunctionalized using n-butylithium and N,NN′,N′-tetramethylethylendiamine in hexane at 60° C., after which thepolymer tube was bubbled with CO₂. The carboxylic acid loading was about1-20 μmol per tube.

Also larger matrix particles, which advantageously provide ease ofhandling, may be used and may be in contact with or proximity to morethan one memory (i.e., one particle may have a plurality of memories inproximity or linked to it; each memory may programmed with differentdata regarding the matrix particle, linked molecules, synthesis or assayprotocol, etc.). Thus, so-called macro-beads (Rapp Polymere, Tubingen,Germany), which have a diameter of 2 mm when swollen, or other matricesof such size, are also contemplated for use herein. Particles of suchsize can be readily manipulated and the memory can be readilyimpregnated in or on the bead. These beads (available from Rapp) arealso advantageous because of their uniformity in size, which is usefulwhen automating the processes for electronically tagging and assayingthe beads.

The matrices may also include an inert strip, such as apolytetrafluoro-ethylene [TEFLON®] strip or other material to which themolecules or biological particles of interest do not adhere, to aid inhandling the matrix, such as embodiments in which a matrix with memoryand linked molecules or biological particle are introduced into anagar-containing plate for immunoassays or for antibiotic screening.

Selection of the matrices will be governed, at least in part, by theirphysical and chemical properties, such as solubility, functional groups,mechanical stability, surface area swelling propensity, hydrophobic orhydrophilic properties and intended use.

The data storage device with programmable memory may be coated with amaterial, such as a glass or a plastic, that can be further derivatizedand used as the support or it may be encased, partially or completely,in the matrix material, such as during or prior to polymerization of thematerial. Such coating may be performed manually or may be automated.The coating can be effected manually or using instruments designed forcoating such devices. Instruments for this purpose are available Isee,e.g., the Series C3000 systems for dipping available from SpecialtyCoating Systems, Inc., Indianapolis, Ind.; and the Series CM 2000systems for spray coating available from Integrated Technologies, Inc.Acushnet, Mass.].

The data storage device with memory may be physically inserted into thematrix material or particle. It also can be manufactured with a coatingthat is suitable for use as a matrix or that includes regions in thecoating that are suitable for use as a matrix. If the matrix material isa porous membrane, it may be placed inside the membrane. It isunderstood that when the memory device is encased in the matrix orcoated with protective material, such matrix or material must betransparent to the signal used to program the memory for writing orreading data. More than one matrix particle may be linked to each datastorage device.

In some instances, the data storage device with memory is coated with apolymer, which is then treated to contain an appropriate reactive moietyor in some cases the device may be obtained commercially alreadycontaining the reactive moiety, and may thereby serve as the matrixsupport upon which molecules or biological particles are linked.Materials containing reactive surface moieties such as amino silanelinkages, hydroxyl linkages or carboxysilane linkages may be produced bywell established surface chemistry techniques involving silanizationreactions, or the like. Examples of these materials are those havingsurface silicon oxide moieties, covalently linked togamma-aminopropylsilane, and other organic moieties;N-[3-(triethyoxysilyl)-propyl]phthelamic acid; andbis-(2-hydroxyethyl)aminopropyltriethoxysilane. Exemplary of readilyavailable materials containing amino group reactive functionalities,include, but are not limited to, para-aminophenyltriethyoxysilane. Alsoderivatized polystyrenes and other such polymers are well known andreadily available to those of skill in this art (e.g., the TENTAGEL®Resins are available with a multitude of functional groups, and are soldby Rapp Polymere, Tubingen, Germany; see, U.S. Pat. No. 4,908,405 andU.S. Pat. No. 5,292,814; see, also Butz et al. (1994) Peptide Res.7:20-23; Kleine et al. (1994) Immunobiol. 190:53-66].

The data storage device with memory, however, generally should not orcannot be exposed to the reaction solution, and, thus, must be coatedwith at least a thin layer of a glass or ceramic or other protectivecoating that does not interfere with the operation of the device. Theseoperations include electrical conduction across the device andtransmission of remotely transmitted electromagnetic radiation by whichdata are written and read. It is such coating that may also serve as amatrix upon which the molecules or biological particles may be linked.

The data storage devices with memory may be coated either directly orfollowing coating with a ceramic, glass or other material, may then becoated with agarose, which is heated, the devices are dipped into theagarose, and then cooled to about room temperature. The resulting glass,silica, agarose or other coated memory device, may be used as the matrixsupports for chemical syntheses and reactions.

Conventional integrated circuit manufacturing and packaging methodsinclude methods and means for encapsulating integrated circuits toprotect the devices from the environment and to facilitate connection toexternal devices. Also, there are numerous descriptions for thepreparation of semiconductor devices and wires, particularly for use assensors [see, e.g., U.S. Pat. No. 4,933,285; see, also Cass, Ed. (1990)Biosensors A Practical Approach, IRL Press at Oxford University Press,Oxford; chemosensors are sensors that can include a biological orchemical detection system, generally biologically active substances,such as enzymes, antibodies, lectins and hormone receptors, which areimmobilized on the surface of the sensor electrode or in a thin layer onthe sensor electrode; biosensors are sensors that detect biologicalspecies and for purposes herein can be implanted in an animal], whichmeasure electrochemical solution parameters, such as pH. Despitedifferences in the components of biosensors and recording devices usedherein, certain of the methods for coating electrodes and wires in thebiosensor art may be adapted for use herein [see, e.g., U.S. Pat. Nos.5,342,772, 5,389,534, 5,384,028, 5,296,122, 5,334,880, 5,311,039,4,777,019, 5,143,854, 5,200,051, 5,212,050, 5,310,686, 5324,591; see,also Usmani et al., ed. (1994) Diagnostic Biosensor Polymers, ACSSymposium Series No. 556].

It is, however, emphasized that the combinations herein of matrix withmemory are not sensors, which measure external parameters and caninclude electrodes that must be in contact with the solution such thatmolecules in solution directly contact the electrode, and which measuresolution parameters. Data regarding the combination, particularly thelinked or associated biological particle or matrix is written into thememory, and thus records information about itself. Sensors monitor whatis going outside of the device. The combinations herein of matrices withmemories can be enhanced by addition of sensor elements for themeasurement of external conditions, information about the externalconditions can be recorded into the combination's memory.

The combinations herein are matrix materials with recording devices thatcontain data storage units that include remotely programmable memories;the recording devices used in solution must be coated with a materialthat prevents contact between the recording device and the medium, suchas the solution or air or gas [e.g., nitrogen or oxygen or CO₂]. Theinformation is introduced into the memory by addressing the memory torecord information regarding molecules or biological particles linkedthereto. Except in the reaction detecting [verifying] embodiment, inwhich the memory can be encoded upon reaction of a linked molecule orbiological particle, solution parameters are not recorded in the memory.

In certain embodiments herein, the matrices with memories herein,however may be combined with devices or components or biosensors orother such sensor devices and used in connection therewith to monitorsolution or external parameters. For example, the combination may beelectronically or otherwise linked to a biosensor and informationobtained by the biosensor can be encoded in memory, or the combinationcan transmit information to the biosensor or, when used internally in ananimal, to monitor the location of a biosensor or to transmitinformation from the biosensor. For example, transponder memory devicesexemplified herein, include circuitry for measuring and recordingsolution temperature. These transponders can be modified to read andrecord pH, instead of or in addition to temperature. Thus, duringsynthesis or other processing steps of linked or proximate molecules orbiological particles, RF or other EM radiation will be used to encodeinformation in the memory and at the same time pH and/or temperature inthe external solution can be measured and recorded in the memory.

1. Natural Matrix Support Materials

Naturally-occurring supports include, but are not limited to agarose,other polysaccharides, collagen, celluloses and derivatives thereof,glass, silica, and alumina. Methods for isolation, modification andtreatment to render them suitable for use as supports is well known tothose of skill in this art [see, e.g., Hermanson et al. (1992)Immobilized Affinity Ligand Techniques, Academic Press, Inc., SanDiego]. Gels, such as agarose, can be readily adapted for use herein.Natural polymers such as polypeptides, proteins and carbohydrates;metalloids, such as silicon and germanium, that have semiconductiveproperties, as long as they do not interfere with operation of the datastorage device may also be adapted for use herein. Also, metals such asplatinum, gold, nickel, copper, zinc, tin, palladium, silver, again aslong as the combination of the data storage device with memory, matrixsupport with molecule or biological particle does not interfere withoperation of the device with memory, may be adapted for use herein.Other matrices of interest include oxides of the metal and metalloids,such as, but not limited to, Pt—PtO, Si—SiO, Au—AuO, TiO2 and Cu—CuO.Also compound semiconductors, such as lithium niobate, gallium arsenideand indium-phosphide, and nickel-coated mica surfaces, as used inpreparation of molecules for observation in an atomic force microscope[see, e.g., III et al. (1993) Biophys J. 64:919] may be used asmatrices. Methods for preparation of such matrix materials are wellknown.

For example, U.S. Pat. No. 4,175,183 describes a water insolublehydroxyalkylated cross-linked regenerated cellulose and a method for itspreparation. A method of preparing the product using near stoichiometricproportions of reagents is described. Use of the product directly in gelchromatography and as an intermediate in the preparation of ionexchangers is also described.

2. Synthetic Matrices

There are innumerable synthetic matrices and methods for theirpreparation known to those of skill in this art. Synthetic matrices aretypically produced by polymerization of functional matrices, orcopolymerization from two or more monomers of from a synthetic monomerand naturally occurring matrix monomer or polymer, such as agarose.Before such polymers solidify, they are contacted with the data storagedevice with memory, which can be cast into the material or dipped intothe material. Alternatively, after preparation of particles or largersynthetic matrices, the recording device containing the data storageunit(s) can be manually inserted into the matrix material. Again, suchdevices can be pre-coated with glass, ceramic, silica or other suitablematerial.

Synthetic matrices include, but are not limited to: acrylamides,dextran-derivatives and dextran co-polymers, agarose-polyacrylamideblends, other polymers and co-polymers with various functional groups,methacrylate derivatives and co-polymers, polystyrene and polystyrenecopolymers [see, e.g., Merrifield (1964) Biochemistry 3:1385-1390; Berget al. (1990) in Innovation Perspect. Solid Phase Synth. Collect. Pap.,Int. Symp., 1st, Epton, Roger (Ed), pp. 453-459; Berg et al. (1989) inPept., Proc. Eur. Pept. Symp., 20th, Jung, G. et al. (Eds), pp. 196-198;Berg et al. (1989) J. Am. Chem. Soc. 111:8024-8026; Kent et al. (1979)Isr. J. Chem. 17:243-247; Kent et al. (1978) J. Org. Chem. 43:2845-2852;Mitchell et al. (1976) Tetrahedron Lett. 42:3795-3798; U.S. Pat. No.4,507,230; U.S. Pat. No. 4,006,117; and U.S. Pat. No. 5,389,449].Methods for preparation of such matrices are well-known to those ofskill in this art.

Synthetic matrices include those made from polymers and co-polymers suchas polyvinylalcohols, acrylates and acrylic acids such aspoly-ethylene-co-acrylic acid, polyethylene-co-methacrylic acid,polyethylene-co-ethylacrylate, polyethylene-co-methyl acrylate,polypropylene-co-acrylic acid, polypropylene-co-methyl-acrylic acid,polypropylene-co-ethylacrylate, polypropylene-co-methyl acrylate,polyethylene-co-vinyl acetate, poly-propylene-co-vinyl acetate, andthose containing acid anhydride groups such as polyethylene-co-maleicanhydride, polypropylene-co-maleic anhydride and the like. Liposomeshave also been used as solid supports for affinity purifications [Powellet al. (1989) Biotechnol. Bioeng. 33:173].

For example, U.S. Pat. No. 5,403,750, describes the preparation ofpolyurethane-based polymers. U.S. Pat. No. 4,241,537 describes a plantgrowth medium containing a hydrophilic polyurethane gel compositionprepared from chain-extended polyols; random copolymerization ispreferred with up to 50% propylene oxide units so that the prepolymerwill be a liquid at room temperature. U.S. Pat. No. 3,939,123 describeslightly crosslinked polyurethane polymers of isocyanate terminatedprepolymers containing poly(ethyleneoxy) glycols with up to 35% of apoly(propyleneoxy) glycol or a poly(butyleneoxy) glycol. In producingthese polymers, an organic polyamine is used as a crosslinking agent.Other matrices and preparation thereof are described in U.S. Pat. Nos.4,177,038, 4,175,183, 4,439,585, 4,485,227, 4,569,981, 5,092,992,5,334,640, 5,328,603.

U.S. Pat. No. 4,162,355 describes a polymer suitable for use in affinitychromatography, which is a polymer of an aminimide and a vinyl compoundhaving at least one pendant halo-methyl group. An amine ligand, whichaffords sites for binding in affinity chromatography is coupled to thepolymer by reaction with a portion of the pendant halo-methyl groups andthe remainder of the pendant halo-methyl groups are reacted with anamine containing a pendant hydrophilic group. A method of coating asubstrate with this polymer is also described. An exemplary aminimide is1,1-dimethyl-1-(2-hydroxyoctyl)amine methacrylimide and vinyl compoundis a chloromethyl styrene.

U.S. Pat. No. 4,171,412 describes specific matrices based on hydrophilicpolymeric gels, preferably of a macroporous character, which carrycovalently bonded D-amino acids or peptides that contain D-amino acidunits. The basic support is prepared by copolymerization of hydroxyalkylesters or hydroxyalkylamides of acrylic and methacrylic acid withcrosslinking acrylate or methacrylate comonomers are modified by thereaction with diamines, aminoacids or dicarboxylic acids and theresulting carboxyterminal or aminoterminal groups are condensed withD-analogs of aminoacids or peptides. The peptide containing D-aminoacidsalso can be synthesized stepwise on the surface of the carrier.

U.S. Pat. No. 4,178,439 describes a cationic ion exchanger and a methodfor preparation thereof. U.S. Pat. No. 4,180,524 describes chemicalsyntheses on a silica support.

Immobilized Artificial Membranes [IAMs; see, e.g., U.S. Pat. Nos.4,931,498 and 4,927,879] may also be used. IAMs mimic cell membraneenvironments and may be used to bind molecules that preferentiallyassociate with cell membranes [see, e.g., Pidgeon et al. (1990) EnzymeMicrob. Technol. 12:149].

3. Immobilization and Activation

Numerous methods have been developed for the immobilization of proteinsand other biomolecules onto solid or liquid supports [see, e.g., Mosbach(1976) Methods in Enzymology 44; Weetall (1975) Immobilized Enzymes,Antigens, Antibodies, and Peptides; and Kennedy et al. (1983) SolidPhase Biochemistry, Analytical and Synthetic Aspects, Scouten, ed., pp.253-391; see, generally, Affinity Techniques, Enzyme Purification: PartB. Methods in Enzymology, Vol. 34, ed. W. B. Jakoby, M. Wilchek, Acad.Press, N.Y. (1974); Immobilized Biochemicals and AffinityChromatography, Advances in Experimental Medicine and Biology, vol. 42,ed. R. Dunlap, Plenum Press, N.Y. (1974)].

Among the most commonly used methods are absorption and adsorption orcovalent binding to the support, either directly or via a linker, suchas the numerous disulfide linkages, thioether bonds, hindered disulfidebonds, and covalent bonds between free reactive groups, such as amineand thiol groups, known to those of skill in art [see, e.g., the PIERCECATALOG, ImmunoTechnology Catalog & Handbook, 1992-1993, which describesthe preparation of and use of such reagents and provides a commercialsource for such reagents; and Wong (1993) Chemistry of ProteinConjugation and Cross Linking, CRC Press; see, also DeWitt et al. (1993)Proc. Natl. Acad. Sci. U.S.A. 90:6909; Zuckermann et al. (1992) J. Am.Chem. Soc. 114:10646; Kurth et al. (1994) J. Am. Chem. Soc. 116:2661;Ellman et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:4708; Sucholeiki(1994) Tetrahedron Lttrs. 35:7307; and Su-Sun Wang (1976) J. Org. Chem.41:3258; Padwa et al. (1971) J. Org. Chem. 41:3550 and Vedejs et al.(1984) J. Org. Chem. 49:575, which describe photosensitive linkers].

To effect immobilization, a solution of the protein or other biomoleculeis contacted with a support material such as alumina, carbon, anion-exchange resin, cellulose, glass or a ceramic. Fluorocarbon polymershave been used as supports to which biomolecules have been attached byadsorption [see, U.S. Pat. No. 3,843,443; Published International PCTApplication WO/86 03840].

A large variety of methods are known for attaching biological molecules,including proteins and nucleic acids, molecules to solid supports [see.e.g., U.S. Pat. No. 5,451,683]. For example, U.S. Pat. No. 4,681,870describes a method for introducing free amino or carboxyl groups onto asilica matrix. These groups may subsequently be covalently linked toother groups, such as a protein or other anti-ligand, in the presence ofa carbodiimide. Alternatively, a silica matrix may be activated bytreatment with a cyanogen halide under alkaline conditions. Theanti-ligand is covalently attached to the surface upon addition to theactivated surface. Another method involves modification of a polymersurface through the successive application of multiple layers of biotin,avidin and extenders [see, e.g., U.S. Pat. No. 4,282,287]; other methodsinvolve photoactivation in which a polypeptide chain is attached to asolid substrate by incorporating a light-sensitive unnatural amino acidgroup into the polypeptide chain and exposing the product to low-energyultraviolet light [see, e.g., U.S. Pat. No. 4,762,8811].Oligonucleotides have also been attached using a photochemically activereagents, such as a psoralen compound, and a coupling agent, whichattaches the photoreagent to the substrate [see, e.g., U.S. Pat. No.4,542,102 and U.S. Pat. No. 4,562,157]. Photoactivation of thephotoreagent binds a nucleic acid molecule to the substrate to give asurface-bound probe.

Covalent binding of the protein or other biomolecule or organic moleculeor biological particle to chemically activated solid matrix supportssuch as glass, synthetic polymers, and cross-linked polysaccharides is amore frequently used immobilization technique. The molecule orbiological particle may be directly linked to the matrix support orlinked via linker, such as a metal [see, e.g., U.S. Pat. No. 4,179,402;and Smith et al. (1992) Methods: A Companion to Methods in Enz.4:73-78]. An example of this method is the cyanogen bromide activationof polysaccharide supports, such as agarose. The use of perfluorocarbonpolymer-based supports for enzyme immobilization and affinitychromatography is described in U.S. Pat. No. 4,885,250]. In this methodthe biomolecule is first modified by reaction with a perfluoroalkylatingagent such as perfluorooctylpropylisocyanate described in U.S. Pat. No.4,954,444. Then, the modified protein is adsorbed onto the fluorocarbonsupport to effect immobilization.

The activation and use of matrices are well known and may be effected byany such known methods [see, e.g., Hermanson et al. (1992) ImmobilizedAffinity Ligand Techniques, Academic Press, Inc., San Diego]. Forexample, the coupling of the amino acids may be accomplished bytechniques familiar to those in the art and provided, for example, inStewart and Young, 1984, Solid Phase Synthesis, Second Edition, PierceChemical Co., Rockford.

Molecules may also be attached to matrices through kinetically inertmetal ion linkages, such as Co(III), using, for example, native metalbinding sites on the molecules, such as IgG binding sequences, orgenetically modified proteins that bind metal ions [see, e.g., Smith etal. (1992) Methods: A Companion to Methods in Enzymology 4, 73 (1992);III et al. (1993) Biophys J. 64:919; Loetscher et al. (1992) J.Chromatography 595:113-199; U.S. Pat. No. 5,443,816; Hale (1995)Analytical Biochem. 231:46-49].

Other suitable methods for linking molecules and biological particles tosolid supports are well known to those of skill in this art [see, e.g.,U.S. Pat. No. 5,416,193]. These linkers include linkers that aresuitable for chemically linking molecules, such as proteins and nucleicacid, to supports include, but are not limited to, disulfide bonds,thioether bonds, hindered disulfide bonds, and covalent bonds betweenfree reactive groups, such as amine and thiol groups. These bonds can beproduced using heterobifunctional reagents to produce reactive thiolgroups on one or both of the moieties and then reacting the thiol groupson one moiety with reactive thiol groups or amine groups to whichreactive maleimido groups or thiol groups can be attached on the other.Other linkers include, acid cleavable linkers, such asbismaleimideothoxy propane, acid labile-transferrin conjugates andadipic acid diihydrazide, that would be cleaved in more acidicintracellular compartments; cross linkers that are cleaved upon exposureto UV or visible light and linkers, such as the various domains, such asC_(H)1, C_(H)2, and C_(H)3, from the constant region of human IgG₁ (see,Batra et al. (1993) Molecular Immunol. 30:379-386).

Presently preferred linkages are direct linkages effected by adsorbingthe molecule or biological particle to the surface of the matrix. Otherpreferred linkages are photocleavable linkages that can be activated byexposure to light [see, e.g., Baldwin et al. (1995) J. Am. Chem. Soc.117:5588; Goldmacher et al. (1992) Bioconj. Chem. 3:104-107]. Thephotocleavable linker is selected such that the cleaving wavelength thatdoes not damage linked moieties. Photocleavable linkers are linkers thatare cleaved upon exposure to light [see, e.g., Hazum et al. (1981) inPept., Proc. Eur. Pept. Symp., 16th, Brunfeldt, K (Ed, pp. 105-110,which describes the use of a nitrobenzyl group as a photocleavableprotective group for cysteine; Yen et al. (1989) Makromol. Chem190:69-82, which describes water soluble photocleavable copolymers,including hydroxypropylmethacrylamide copolymer, glycine copolymer,fluorescein copolymer and methylrhodamine copolymer; Goldmacher et al.(1992) Bioconj. Chem. 3:104-107, which describes a cross-linker andreagent that undergoes photolytic degradation upon exposure to near UVlight (350 nm); and Senter et al. (1985) Photochem. Photobiol42:231-237, which describes nitrobenzyloxycarbonyl chloride crosslinking reagents that produce photocleavable linkages]. Other linkersinclude fluoride labile linkers [see, e.g., Rodolph et al. (1995) J. Am.Chem. Soc. 117:5712], and acid labile linkers [see, e.g., Kick et al.(1995) J. Med. Chem. 38:1427]. The selected linker will depend upon theparticular application and, if needed, may be empirically selected.

B. Optically encoded memory devices

The matrices or strips attached thereto may be encoded with apre-programmed identifying bar code, such as an optical bar code thatwill be encoded on the matrix and read by laser. Such pre-coded devicesmay be used in embodiments in which parameters, such as location in anautomated synthesizer, are monitored. The identity of a product orreactant determined by its location or path, which is monitored byreading the chip or memory in each device and storing such informationin a remote computer.

Thus, it is contemplated herein, that the memory is not proximate to thematrix, but is separate, such a memory in a remote computer or otherrecording device. In these embodiments, the matrices are marked with aunique code or mark of any sort. The identity of each mark is saved inthe remote memory, and then, each time something is done to a moleculeor biological particle linked to each matrix, the information regardingsuch event is recorded and associated with the coded identity. Aftercompletion of, for example, a synthetic protocol, each matrix isexamined or read to identify the code. Retrieving information that fromthe remote memory that is stored with the identifying code will permitidentification or retrieval of any other saved information regarding thematrix.

For example, simple codes, including bar codes, alphanumeric charactersor other visually or identifiable codes or marks on matrices are alsocontemplated for use herein. When bar codes or other precoded devicesare used, the information can be written to an associated but remotememory, such as a computer or even a piece of paper. The computer storesthe bar code that a identifies a matrix particle or other code andinformation relating to the molecule or biological particle linked tothe matrix or other relevant information regarding the linked materialsor synthesis or assay. Instead of writing to an on-board memory,information is encoded in a remote memory that stores informationregarding the precoded identity of each matrix with bar code and linkedmolecules or biological particles. Thus, the precoded information isassociated with, for example, the identity of the linked molecule or acomponent thereof, or a position (such as X-Y coordinates in a grid).This information is transmitted to a memory for later retrieval. Eachtreatment or synthetic step that is performed on the linked molecule orbiological particle is transmitted to the remote memory and associatedwith the precoded ID.

For example, an amino acid is linked to a matrix particle that isencoded with or marked with a bar code or even a letter such as “A” orother coded mark. The identity the amino acid linked to the matrixparticle “A” is recorded into a memory. This particle is mixed withother particles, each with a unique identifier or mark, and this mixtureis then treated to a synthetic step. Each particle is individuallyscanned or viewed to see what mark is on each particle and the remotememory is written to describe the synthetic step, which is thenassociated with each unique identifier in the memory, such as thecomputer or piece of paper. Thus, in the remote memory the originalamino acid linked to particle A is stored. After the synthetic step, theidentify of the next amino acid is stored in the memory associated with“A” as is the identity of the next amino acid added. At the end of thesynthesis, the history of each particle can be read by scanning theparticle or visually looking at the particle and noting its bar code ormark, such as A. The remote memory is then queried to determine whatamino acids are linked to the particle identified as “A” [see, e.g.,FIG. 20].

For example, many combinatorial libraries contain a relatively smallnumber of discrete compounds [10²⁻¹⁰ ⁴] in a conveniently manipulablequantity, rather than millions of members in minute quantities. Thesesmall libraries are ideal for use with the methods and matrices withmemories herein. They may also be used in methods in which the memory isnot in proximity to the matrix, but is a remote memory, such as acomputer or a table of information stored even on paper. The systemdepicted in FIG. 20 is ideal for use in these methods. Polypropylene orother inert polymer, including fluoropolymers or scintillating polymersare molded into a convenient geometry and size, such an approximately 5mm×5 mm×5 mm cube [or smaller or larger] with a unique identifying codeimprinted, preferably permanently, on one side of each cube. If, forexample, a three element code is used, based on all digits (0to 9) andall letters of the alphabet, a collection of 46,666 unique three elementcodes are available for imprinting on the cubes.

The cubes are surface grafted with a selected monomer [or mixture ofmonomer], such as styrene. Functionalization of the resulting polymerprovides a relatively large surface area for chemical syntheses andsubsequent assaying [on a single platform]. For example, a 5×5×5 mm³cube has a surface area of 150 mm², which is equivalent to about 2-5μmol achievable loading, which is about 1-2.5 mg of compounds with amolecular weight of about 500. A computer program, described below [see,e.g., Examples], or protocol can direct split and pool during synthesisand the information regarding each building block of the linkedmolecules on each cube conveniently recorded in the memory [i.e.,computer] at each step in the synthesis.

Since the cubes [herein called MACROCUBES™ or MACROBEADS™] arerelatively large, they can be read by the eye or any suitable deviceduring synthesis and the associated data can be manually entered into acomputer or even written down. The cubes can include scintillant orfluorophore or label and used in any of the assay formats describedherein or otherwise known to those of skill in the art.

For example, with reference to FIG. 20, polypropylene, polyethylene orfluophore raw material [any such material described herein, particularlythe Moplen resin e.g., V29G PP resin from Montell, Newark Del., adistributor for Himont, Italy] 1 is molded, preferably into a cube,preferably about 5×5×5 mm³ and engraved, using any suitable imprintingmethod, with a code, preferably a three element alphanumeric code, onone side. The cube can be weighted or molded so that it all cubes willorient in the same direction. The engraved cubes 2 are thensurface-grafted 3 and functionalized using methods described herein orknown to those of skill in this art, to produce cubes [MACROBEADS™ orMACROCUBES™] or devices any selected geometry 4.

1. Encoded Memory Devices With Two-dimensional Bar Codes a. MatricesWith Optical Memories

In another exemplary embodiment, illustrated in FIG. 22, the opticalmemory device [“OMD”] 100 is a preferably a rectangular parallelepipedthat provides a broad face upon which encoded information can beinscribed. Any geometry that is suitable for a particular applicationand that provides at least one surface for encoding information. TheOMDs may also be containers used for chemical synthesis, such asmicrotiter plates, tubes, tubes adapted for use with microtiter-typeplates. The two-dimensional bar code described herein is ideally suitedfor incorporation onto the outside surface of each well of a microtiterplate or on the outside of a small test tube or other such tube,particularly, tubes intended for use with a microplate frame, such asthose available from NUNC and COSTAR. This two-dimensional bar code aswells as the method for reading and writing may also be used to trackand identify other laboratory equipment, such as chromatography tubes,test tubes, beakers, flasks and other such items.

The OMDs may also be fabricated as tubes, such as the MICROTUBES™provided herein. When used with such tubular devices, they will beengraved on the outer surface, preferably the top or bottom of thedevice.

The material of which the OMDs are fabricated will depend upon themonitored processes. The materials that may be used include, but are notlimited to, black, white or colored glass, TEFLON®, polyethylene, highdensity polyethylene, polypropylene, polystyrene, polyester, ceramic,such as alumina or zirconia, metal, or any composite of the abovematerials or any material that is physically or chemically structured toproduce optical contrast as the result of exposure to the write process,which is described below. For use in the methods herein, these materialsmay be suitable or at least one surface there may have been treated torender them suitable for retaining molecules and biological particlesfor use as matrices as described herein.

For the first exemplary embodiment of OMD 100 shown in FIG. 22, if theOMD is formed from a ceramic material, it may have exemplary dimensionsof 280 mil (L)×140 mil (W)×50 mil (T) [7 mm×3.5 mm×1.3 mm]. Thedimensions of the face can be varied as needed to provide theappropriate size for recording data, providing sufficient chemicalbinding surface area, and to facilitate handling. The presentlypreferred minimum size for use with commercial feeding systems is on theorder of 0.5 mm×0.5 mm×0.5 mm.

If the OMD 100 is formed from polypropylene, it may have exemplarydimensions of 280 mil (L)×140 mil (W)×100 mil (T) [7 mm×3.5 mm×2.6 mm],although smaller dimensions are contemplated. Since OMDs made frompolypropylene may be read by transmission of light through the device,the thickness must be sufficiently thin to permit transmission of lightthrough the OMD, except where there are darkened areas of a bar codesymbol. Where reflected light is to be used, as with the ceramic OMDs,thickness need not be so limited.

For OMDs used for chemical binding or other processes for which surfacesmust be specially prepared in order to assure adsorption or absorptionor any means of binding of molecules or biological particles, it may bedesirable to separate the binding surfaces from the data storage surface101. In this case, one or more of sides 104 and 105, bottom 107, top108, and back 110 may be treated to enhance binding using radiation,mechanical or chemical abrasion, or other processes as appropriate. Bysegregation of the binding and information surfaces, possible activationor modification of certain bound compounds by the high intensity lightsource used in the write process is avoided. In addition, degradation ofthe bar code contrast may be less on a surface that is not derivatizedfor binding.

If needed, segregation of the binding and information surfaces can beachieved by coating portions of the OMD with films formed from adielectric material such as polyethylene, MYLAR, TEFLON®, KAPTON,polycarbonate, or, preferably, the para-xylylene polymers sold under thetrade name Parylene [see, e.g., U.S. Pat. Nos. 3,288,728, 3,342,754 and3,429,739], or any other such materials that are commonly used in theelectronics industry to passivate electronic components and circuitboards, and as a coating for medical devices, especially implants,catheters, probes and needles. [Parylene is the trade name for membersof a series of polymers which are commercially available from SpecialtyCoating Systems, Inc., of Indianapolis, Ind. and originally from UnionCarbide Corporation, Greenville, S.C., see, U.S. Pat. Nos. 3,288,728,3,342,754 and Gorham 3,429,739; see, also brochures distributed by themanufacturer, entitled “Parylene Conformal Coatings Specifications andProperties” (©1984, Specialty Coating Systems, Inc.), and “Parylene, ABiostable Coating for Medical Applications” (©1984, Specialty CoatingSystems, Inc.]. These polymers provide a conformal biostable coatingwhich electrically and chemically isolates the protected surface fromits environment.

The Parylene or other such polymeric coating can be treated to form achemically functional substrate by methods such as beta or gammaradiation, and mechanical or chemical roughening. Alternatively,polystyrene microspheres can be bonded [glued or welded] to selectedsurface(s) of the OMD, either on the Parylene or similar coating, ordirectly to the ceramic or polypropylene.

The encoded information may be stored in any optically writable andreadable format. As shown printed on data storage surface 101,symbologies 106 are two-dimensional bar codes, which can be stacked rowsof one-dimensional bar codes, checkerboards, or dot matrices. Othersymbologies that can be used include one-dimensional bar codes, targetcodes, alphanumeric characters or other optically readable characterswhich are well known in the art. [See, e.g., Wang, et al. (1990) A HighDensity Two Dimensional Bar Code SPIE Proceedings Vol. 1384, High-SpeedInspection Architectures, Bar Coding, and Character Recognition, pp.169-175; Martin (1991) Unique Symbol for Marking and Tracking Very SmallSemiconductor Products, SPIE Proceedings Vol. 1598, Lasers inMicroelectronic Manufacturing, pp. 206-220.]

In the exemplary embodiment, the two-dimensional bar code [e.q., symbol106] includes an orientation indicator in the form of solid black linesacross the top 120 and down the right side 122 of the symbol. Uponacquisition of the image of the symbol by the image sensing means, theimage processor will utilize the orientation indicator to provideinformation about the rotation of the OMD relative to the sensor, andcan compensate in its software by rotating the image to the appropriateorientation for decoding the image. Other types of orientationindicators as are known in the art, such as those described in theabove-identified references relating to bar codes, may also be used suchthat physical precise orientation of the OMD within the read area is notcritical. For reflection-type readers, it is only necessary for the OMDto be right side up and the symbol is fully within the field of view ofthe detector, so that the symbol 106 is exposed to the image sensor.Even where reading is accomplished by transmission of light through theOMD, as in certain polypropylene embodiments, an orientation indicatorin the symbol in combination with a distinctive physical or opticalfeature, such as described below, can provide information sufficient todetermine whether the OMD is face up or face down so that appropriatecompensation, such as reversal of the image, can be performed by thesoftware in order to enable decoding.

An alternative means for recording and reading information involves theformation of a magnetic film on at least a portion of the surface of theOMD. Creation of thin magnetic films by sputtering, electroplating, orother deposition techniques are well known in magnetic recordingtechnology. [See, e.g., Chapter 11, “Tape and Disk Materials” from TheComplete Handbook of Magnetic Recording, 3rd Edition, by Finn Jorgenson,Tab Books, 1988.] Recording and reading of data on the magnetic film canutilize conventional magnetic recording techniques.

The OMD 200 of FIG. 23 is a variation on the embodiment of FIG. 22 thatprovides a information recording section that is formed from a separatematerial from that of the binding surface(s) [i.e., the chemistrysurface(s) or the surface(s) to which molecules or biological particlesare linked]. In this embodiment, the OMD contains two sections that arelinked together. Here, OMD 200 is formed from the assembly ofinformation unit 202 and binding unit 204, with unit 202 fitting withina cavity or well 206 formed in unit 204. This embodiment provides theadvantage of selecting the optimal material for each of the binding andrecording processes, and also permits the information unit 202 to beassembled with the binding unit 204 after the binding unit has beentreated to enhance adhesion. For example, binding unit 204 can be formedfrom a polymer, e.g., polypropylene, functionalized by radiation and/orchemical processes, or can be modified by bonding polystyrenemicrospheres to its surface(s). Information unit 202 can be formed fromplastic, ceramic or glass, and mounted within well 206 by adhesive orother bonding process, or may simply be press fit into the well. Sincepre-treatment of the binding unit to enhance binding could possiblydiscolor the information unit, or otherwise make it less readable bymodifying the surface, e.g., pitting or etching, separate formationcould be advantageous. The outer dimensions of unit 202 are preferablyselected to closely fit the inside dimensions of well 206 to prevent theintrusion of chemicals, or even air, into spaces between the units. Inthe illustrated example, unit 204 has outer dimensions of 280 mil(L)×140 mil (W)×100 mil (T) [7 mm×3.5 mm×2.6 mm] and unit 202 hasmaximum dimensions of 210 mil (L)×115 mil (W)×50 mil (T) [5.3 mm×2.9mm×1.3 mm]. Since, as shown, the sides of unit 202 are beveled to form atrapezoidal cross-section to conform to a corresponding shape of thewell 206, and also to assist in forming a tight seal between the twounits, the actual exposed face of the information unit is on the orderof 105 mil×200 mil [2.7 mm×5 mm]. When the two units are assembled, thecombined face surface of the information and binding units arepreferably flush. As can be seen, the encoded information, shown as atwo-dimensional bar code symbol 208, is inscribed on information unit202 only. With regard to the magnetic recording alternative method, theuse of a separate information unit is ideal since it would generally bepreferred to avoid exposure of magnetic recording media to the radiationor corrosive chemicals used for enhancement of the binding process.

Variations on the two-part OMD of FIG. 23 are illustrated in FIGS.25-27. In FIG. 25, OMD 400 is illustrated where insert unit 402 is thebinding unit formed, for example, from polymer functionalized byradiation and/or derivatized by suitable chemical processes or graftedto render the surface suitable for binding biological particles andmolecules. Base unit 404, which may be formed from plastic, polymer,ceramic or glass, has a well 406 corresponding to the exterior shape ofthe binding unit 402, so that they will interfit closely. The encodedinformation, shown, again, as a two-dimensional bar code symbol 412, isinscribed on the back 408 of base unit 404, opposite the face 410 atwhich binding unit 402 is exposed.

In FIG. 26 [an embodiment of a microvessel], insert unit 502 has acavity 508 covered by mesh 510 [porous material] for retaining particlesbut permitting chemical materials and biological particles to passthrough, to form OMD 500. The chemicals pass through mesh 510 to bewithin cavity 508, or some material contained therein, such asmicrospheres, or are retained on the strands of mesh 510. As in theembodiment of FIG. 25, base unit 504, which is encoded with thesymbology, receives the binding [chemistry] unit so that it is exposedon one face 512, with the encoded information 514 located on theopposite face 516.

In the embodiment of FIG. 27, insert unit 602 is formed frompolypropylene or ceramic or other suitable material and provides theinformation storage face 608 for writing symbology, preferably a barcode symbol 610 on OMD 600. Base unit 604 provides the means for bindingof chemical materials, which contains cavity 612 which is filled withmicrospheres 614 and covered with polypropylene screen 616 or othersuitable porous material. The base material is preferably polypropyleneor other such material. In this embodiment, the information storage face608 is on the opposite side of the OMD from the screen 616.

In yet another embodiment, OMD 700, which is illustrated in FIG. 28, anorientation indicator is provided in the form of a notched or cut-corner702. In this embodiment, the corner cut-out 702 will provide informationas to the rotation and inversion of OMD 700, since, even if the OMD isface down, it will be apparent due to the unique outline of the face.The use of a physically detectable orientation indicator allows thehandling equipment to readily detect improper positioning, for example,by placement of mechanical or optical edge detectors within the handlingsystem. An improperly positioned OMD can be removed from the imagingposition and placed back at the entry point into the reading handler, ormechanical means, such as a retractable blade, can be provided to flipthe OMD over if it is presented face down within the field of view ofthe reader. An alternative symbology 706 is illustrated which is, inthis case, an alphanumeric code, which can be read and decoded usingknown optical character recognition (OCR) techniques.

Other types of orientation indicators that can be used include chamfers,holes and protrusions. Several different and distinctive shapes can beincluded on a single OMD to assist in orientation, positioning andseparation of the OMDs. For example, a group of OMDs can have a cutcorner for orientation of each OMD, with some of those OMDs having a tabextending from one of its sides, so that those with tabs can beseparated from those without tabs, which facilitates division of thegroup for diversion to different containers.

Additional test media can be included in the OMD in the embodiment ofFIG. 29. Here, the OMD 800 has a plurality of wells or recesses 804 intowhich can be placed gels, beads, or the like for retaining additionalchemical [molecules] or biological materials [biological particles],and/or chemical, biological or temperature sensors, or other suchdevices. Where such materials are placed in the wells 804, the bar codesymbol 806 can include information about the nature of these materials.

The embodiment of FIG. 30 is a variation on that of FIG. 29. Here, theOMD 900 is partially hollow, and a plug 902 is formed in the side topermit access to the cavity 912. The front 904 and/or back 908 walls ofthe OMD have a mesh insert 910 which provides limited access to thecavity 912 in the OMD. A chemically- or biologically-functional material[biolgocial particle or molecule], or microspheres, for example, can beplaced within the cavity 912 through plug 902 so that it is exposed tothe chemical or biological materials to which the OMD is exposed withoutallowing direct contact between the material in the cavity and theenvironment in which the OMD is placed. The mesh [porous material] 910can be polypropylene or other such suitable polymer, and of a size thatmakes it semi-permeable, admitting the external solution withoutallowing the interior material to escape. Generally, the pore size willbe within the range of 20 μm to 200 μm. The use of OMDs and protocolstherefor are flexible and can employ a variety of shapes,configurations, and polymeric synthesis supports. The ceramic 2-D barcodes will have at least 8-bytes of information content. When etched onceramics, they are inert to the vast majority of organic synthesisconditions, easy and reliable to use, inexpensive, and very amenable tomass production. The low cross-linking polystyrene surface graft (andother polymer grafts) on the stable and inert base polymer provides anideal solid support for chemical synthesis with excellentfunctionalizability and chemokinetics. This technology can also beapplied to the synthesis of other types of compounds, especially smallorganic molecules. Among the advantages of this technology over existingcombinatorial techniques are: a) low manufacturing cost; b) non-invasiveencoding; c) high encoding reliability and capacity; d) total chemistryflexibility; e) excellent chemokinetics; f) easy and clean washingbetween reactions; g) utilization of the highly efficient directedsorting strategy; h) delivery of pure, discrete compounds inmulti-milligram scale; and i) very amenable to full automation.Integration of the laser optical synthesis OMD technology withautomation will further enhance its applications in high through-putchemical synthesis and biological screening.

These devices have a wide variety of applications. For example, withreference to FIG. 33E, the bar-coded OMDs devices are functionalizedwith amino groups to give functionalized OMDs devices 3 and used in thesynthesis of oligomers. The first set of nucleosides modified with asuccinic acid linker are coupled onto the matrices using DNA synthesizerwith modified reaction vessels [suitable for use with the OMD-linkednascent oligonucleotides]. Five cycles of TCA de-blocking, sorting,tetrazole activation, coupling, capping, and oxidation are performedautomatically on the machine [except the sorting] to yieldoligonucleotides [hexamers] on the matrices 6. Cleavage and deprotectionunder standard conditions give the oligonucleotide hexamer library 7.The identity of each oligonucleotide is associated with the unique codein a remote memory, such as by manually entry before, during or aftersynthesis.

b. Reading and Writing to Matrices With Optical Memories

An exemplary read/write system is illustrated in FIG. 24. The writesystem includes laser 320, mirror 322, and prism 324 mounted on driveshaft 325 connected at a first end to drive motor 326. Drive shaft 325is connected at its second end to geared linkage 328 which rotates driveshaft 329 and prism 330 in synchrony with prism 324. The beam emitted bylaser 320 follows optical path 310 to mirror 322, where it is reflectedtoward prism 324. Prism 324 rotates to scan the beam along the y-axis,i.e., up and down, so that the beam effectively shifts top to bottom byreflection from the prism faces in succession as it rotates. This beamis similarly scanned along the x-axis from left to right by reflectionfrom the faces of prism 330 in succession as prism 330 rotates. Eitheror both prisms can be replaced with rotating or oscillating mirrors toachieve the same scanning pattern, in a manner similar to the scanningmechanisms used in conventional laser-based bar code scanners [see,e.g., U.S. Pat. No. 4,387,297 to Swartz, et al., entitled “PortableLaser Scanning System and Scanning Methods”, and U. S. Pat. No.4,409,470 to Shepard, et al., entitled “Narrow Bodied, Single- andTwin-Windowed Portable Laser Scanning Head for Reading Bar CodeSymbols”]. Mirrors 332 and 334 provide means for directing the beamtoward the OMD 300 at the appropriate level, and, thus, are positionedin consideration of the guide means, so that the beam impinges upon thedesired recording surface.

As illustrated in FIG. 24, OMD 300 has already been inscribed during anearlier process step, evidenced by the fact that symbol 306 is presentand complete. Also as illustrated, symbol 316 is currently being writtenby the progression of laser spot 336 across the write surface, asscanned by prisms 324 and 330. The contrasting dark and light areas ofthe symbols 306,316 are created by pulsing the laser 320 according to asignal provided by system controller 346.

In an exemplary embodiment, laser 320 is a CO₂ laser, which emits lightin the infrared at a wavelength of 10.6 μm. The writing process isaccomplished by using a sufficiently high power beam to burn the surfaceof the OMD, formed of ceramic, white polypropylene or the like, toproduce a dark carbon build-up corresponding to the dark lines of thesymbology on the lighter colored background. The exemplary laser poweris 25 W, with a spot size of 0.03 mm, burning a dot in the write surfaceof 0.13 mm, to create a two-dimensional bar code using a dot matrixpattern. Mirrors 322, 332 and 334, and prisms 324 and 330 must be coatedwith an appropriate IR-reflective film to avoid damage to the optics bythe laser. In read systems which utilize transmission of light throughthe OMD, the carbon build-up will block the light, appearing as darkenedareas to a sensor on the opposite side of the OMD from the light source.In read systems which utilize back-reflection, the carbon build-up willabsorb light, while the other areas will reflect the light, againcreating a contrast between the inscribed and untouched areas of thesurface.

Since the IR write beam is not visible, it may be desirable to use anoptically-visible laser (not shown), for example, He—Ne or a diode laserwhich emits within the visible spectrum, or other focused light source,to emit a beam along optical path 310 to permit visual alignment. Theoptically-visible laser may also be used as a proximity detector to senda signal to the system controller to indicate the presence of the OMD inthe write position to trigger the write process, either by reflection orby blocked transmission using conventional optical sensors.Alternatively, a separate optical detector system may also be used todetect and indicate the presence of an OMD in the write or readposition.

For OMDs formed from glass or ceramic, a beam from a CO₂ laser can beused to etch the glass to produce contrasting lines by modifying thesurface finish of the glass. For example, the glass can be frosted orotherwise roughened, which may assist in the binding of compounds, andwhich has a reduced reflectivity. Upon exposure to the high power writebeam, the glass surface is partly flowed, i.e., partly melted, so that asmooth, highly reflective surface remains after the surface cools.Contrast between the frosted and flowed glass can be enhanced forreading by selecting a read wavelength which maximizes the differencesin reflectivity between the two surface finishes.

Other types of lasers which may be used include neodymium-YAG[yttrium-aluminum-garnet], excimer, or any other laser capable ofemitting a sufficiently high power beam to modify the material surfaceto produce an optically-readable contrast. Alternative lasers for thewriting process include diode lasers, such as those made by Coherent,Inc. of Santa Barbara, Calif. Among those that are suitable are ModelNo. S-98-2000C-200-C, T or H, which emit light at 980 nm with a CW powerof 2000 mW, Model No. B1-81-10C-19-30-A or W, which emit light at 808 nmwith a CW power of 10,000 mW, or Model No. B1-81-15C-19-30-A or W, whichemit light at 808 nm with a CW power of 15,000 mW.

Selection of the laser will depend on the material of which the OMDs areformed. For example, if an optically-reactive material is encased withina transparent glass or plastic shell, any laser capable of inducing thereadable change in the optically-reactive material would be acceptable.Photochemically-reactive media, known to those of skill in the art [see,e.g., U.S. Pat. No. 5,136,572 to Bradley, entitled “Optical Data StorageSystem”] can be selectively activated and read by use of wavelengthtunable diode lasers, such that a lesser power and/or visible lightlaser can be used with a more reactive recording media.

The range of movement of the laser spot 336 is limited, generally to thearea of the write surface, so that the OMDs must be moved past a targetarea within which laser spot 336 is projected. Movement of the OMDs canbe achieved by one or more sets of conveyor belts, chutes or guiderollers, each of which can be fed by a commercial-type centrifugalfeeder, such as those available from Hoppmann Corporation of Chantilly,Va. and Kirchlintein, Germany. Feeders of this type are known inindustry for mass handling of parts and products, including foods,pharmaceuticals, containers and hardware. Linear and vibratory feedersare also known and may be used for handling the OMDs. An exemplaryhandling system is illustrated in FIG. 32 and will be discussed in moredetail below.

Included in the proximity location process to detect the presence of anOMD within the write position can be a detector 372 for locating thenext available area on the write surface for writing. In the exampleillustrated in FIG. 24, for a write surface having four availablelocations, position #1 is already filled with symbology 306, andposition #2 is in the process of being filled with symbology 316. Alight source 368, such as a visible laser, emits a beam which isdirected by scanning optics 370 toward the write surface of the OMD. Asthe write surface is scanned by the beam, the next available areadetector 372 can look sequentially at each OMD as its presence isdetected, first at position #1, then at subsequent positions until itfinds an area on which no symbology is written, i.e., no contrastingmarkings are detected, or a “white” area of a pre-determined width isdetected which is wider than the “quiet Zone” which is commonly includedin bar codes. [See, e.g., Wang, et al., “A High Density Two DimensionalBar Code,” SPIE Proceedings Vol. 1384, High-Speed InspectionArchitectures, Bar Coding, and Character Recognition (1990) pp.169-175.] This location process permits multiple uses of OMDs, and takesinto consideration that some OMDs may be exposed to a greater number ofprocess steps than others before being combined into the string in whichthey are presently included.

The detector 372 can also be used for indication of the presence of OMDsto be read. In the case of reading, the detector 372 can also be used toidentify the presence of all symbologies to be scanned for reading,which is particularly important if a laser beam or other relativelynarrow beam of light is scanned over the written surface to read thesymbology. Where an incoherent light source is used to simply flood theentire write surface with light, such as a lamp 340, the ability todetect the presence of individual symbologies is not critical, since theentire write surface will be viewed and recorded at once using framegrabbing techniques.

During the read process, after the presence of an OMD is indicated, thelamp 340 is activated to illuminate the write surface. The lightreflected from the surface is modulated by the symbology printed thereondue to the selective reflection and absorption of the contrasting areas.Optics 342, which will typically be an assembly of lenses and filters,which remove stray light, focus the reflected light onto detector 344.Selection of optics can be performed in a using methods known to thoseof skill in the art [see, e.g., U.S. Pat. No. 5,354,977 of Roustaei,which describes an optical bar code scanner using a CCD [charge-coupleddevice] detector and associated optics. In the same reference, adetailed description of CCD detectors for use in bar code scanners isprovided, as well as steps for processing the signal generated by theCCD detector.

In the exemplary embodiment illustrated in FIG. 24, the CCD detector 344comprises an array of discrete devices, each of which is a “pixel”,capable of storing charge impinging upon it representative of reflectedlight from the write surface, then reading out the charge as a serialanalog waveform. A typical CCD array for bar code scanning has 2048pixels, however, CCD arrays of other dimensions may be used. In thepreferred embodiment, a CCD array of 640×480 pixels is used. Using theCCD array, a “snap shot” of the OMD surface is created using known imageor frame grabbing techniques, and an analog electrical representative ofthe snap shot is conducted to the signal processing function 348 withinthe system controller 346, which includes an analog-to-digitalconverter, to convert the signal into an output of the data written onthe OMD.

In a preferred embodiment, the detector is a commercially-available,PC-interfaceable CCD camera sold under the trademark QuickCam™ byConnectix Corporation of San Mateo, Calif., which has a resolution of640×480 pixels. Any other such camera may be used. The camera has amanually-adjustable focus lens, but image acquisition is otherwisecontrolled by the system controller 346, which, as part of its software,initializes the camera for frame grabbing. Any such PC-interfaceable CCDcamera with a similar or better resolution may be used. Other types ofdetector arrays known within the bar code scanning technology, includingCMOS sensors, can be used in place of the CCD detector array 344.

Processing of the image grabbed by the image detector is a significantaspect of the system in that it provides the flexibility to manipulatethe image to enhance readability. The steps of the exemplary imageprocessor are provided in the flow diagram of FIG. 31, and the imagesignal generated by the detector is checked for completeness, validityand orientation, among other things. As discussed above, if systemswhere physical orientation and positioning of the OMD is not assured bythe handling hardware, one aspect of the image processing software is todetermine skew or rotation of the image as seen by the detector. Thefollowing steps are provided in detail in the system processor'ssoftware, and a portion of which is depicted in the flow diagram of FIG.31. [Note that the actual image obtained from the camera can bedisplayed on a system monitor as it is being modified to permitdecoding.] First, after obtaining the image from the camera [step 1001],in steps 1002 and 1003, the edges of the symbol in the verticaldirection are identified, looking for the highest peak signal to providea reference, then the horizontal edges are found [step 1004]. Knowingthe boundaries of the symbol, the reasonable spacing is determined [step1005] to correct for missing or extra vertical edges using a neuralnetwork approach. Based on the reasonable spacing, it is determined ifthe length of the vertical edge is appropriate [step 1006]; if not,adjustments are made by adding or removing edges [step 1007]. A similarprocedure is used for the horizontal edges [steps 1008-1010], allowingskew to be determined. Having determined the orientation and spacing ofthe symbol, the symbol is broken into sections [step 1011], or cells,and the average intensity for each cell is determined [step 1012] topermit calculation of the threshold [step 1013] for distinguishing adark from a light area of the code. Following this, parity checks areperformed [step 1014] to provide an indication of whether the completesymbol was detected, i.e., the correct number of bits was obtained, orwhether the signal was corrupted. In the preferred embodiment, 17 bitsof the data contained within the two-dimensional bar code are dedicatedto parity checking. At this point, if a corrupted signal is indicated,and the error is not corrected [step 1015], rather than proceeding withan attempt to decode, the image processor initiates a new scan of thesymbol [step 1001]. If the signal is good, the cells are converted tocode [step 1016] which is decoded [step 1017].

The image processing system converts the stored image into a series ofrows [and columns for two-dimensional codes] containing binary data,with dark lines indicating highs, or ones, and white lines indicatinglows, or zeroes [or vice versa]. To enhance accuracy, a number ofprocessing steps may be performed, and the resulting data can be theaverage of all processing steps for that particular image.

Although efforts are taken initially to avoid modification of thesurface on which the information is written, because each OMD ispresumably being subjected to a significant number of process steps, theappearance of the symbol may degrade with time due to accumulation ofchemicals or surface roughening, resulting in decreased contrast betweenthe light and dark lines of the code. This issue can be addressed withsoftware, which can compensate for the deterioration of the symbol. In apreferred embodiment, the software includes neural network algorithmswhich can be trained to learn the specific cumulative effects ofchemical processing and compensate by either recalibrating the detector,for example, to increase the exposure time, to modify the illumination,to increase the number of verification decode steps used for averaging,or to adjust the threshold between “dark” and “light”. Two appropriatecommercially-available neural network programs are Thinks™ andThinksPro™ [published by Logical Designs Consulting, Inc. La Jolla,Calif.; see, U.S. Pat. No. 5,371,809], which are designed to run onpersonal computers, and provides numerous different and well knowntraining algorithms and methods. Other neural network software productsare commercially available, including Neural Works Professional™ byNeuralWare, NeuroShell2™ by Ward Systems, BrainMaker Professional™ byCalifornia Scientific, and Neural Net Tool Kit™ by Math Works. Eachcould be used for training of the signal processor to compensate fordegraded contrast in the symbol, and selection of the appropriateprogram or creation of an appropriate program would be within the levelof skill in the art.

FIG. 32 provides a diagram of an exemplary handling system forseparating and reading and/or writing to an OMD, particularly those inthe shape of a parallelopiped. Such handlers are commercially available[e.g., from Hoppmann Corporation, Chantilly, Va., see, U.S. Pat. Nos.5,333,716, 5,236,077, 5,145,051, 4,848,559 4,828,100, 4,821,920,4,723,661 and 4,305,496]. The OMDs are placed in vibratory feeder 1102by way of supply hopper 1104. Vibratory feeder 1102 includes rings andramps [not shown] which support the OMDs as they move within the feeder,driven by the feeder's vibration in a direction toward exit chute 1106.An orientation rim, bar, or other feature [not shown] may be included inthe internal ramps or exit chute to rotate the OMDs when a physicalorientation indicator, such as the cut corner, is provided. Exit chute1106 feeds the OMDs to ramp 1110 of linear feeder 1108. Thereciprocating motion of the ramp 1110 causes the OMDs to move forward[to the left in the figure) toward walking beam 1112 and within thefield of view of camera 1114. [Where a write operation is to beperformed, the write laser and optics can be positioned in place of ornearby the camera.] Movement of the walking beam 1112 is stepped so asto pause advance motion of the OMD to allow writing and/or reading ofthe appropriate information.

After completion of the writing or reading step, the OMD is advancedalong the walking beam 1112 toward one or more vials or flasks 1114containing chemical or biological solutions. Ramps [not shown] leadingfrom the walking beam to the vials or flasks 1114 can be selected byopening gates, or by tilting the walking beam 1112 in front of theselected vial, thus feeding the OMD into the desired vial for the nextprocess step. The vials or flasks 1114 can be fixed within a tray orrack that allows it to be removed after the processing has finished sothat the OMDs can be dumped into the hopper of the same or anotherfeeder to repeat the above steps for handling, writing, reading, anddistributing the OMDs to the next process step.

It may be desirable to include a protective enclosure 1116, such as apolycarbonate and polyphenylene oxide resins, preferably thepolycarbonate resin sold under the name LEXAN™ [the well knownpolycarbonate resin commercially available from General Electric Corp,Waterford, N.Y., or MERLON™ made by Mobey Chemical Co., Pittsburg, Pa.]or the resin sold under the tradename NORYL [from General Electric Corp]other such polymer such as polyethylene, lucite, bakelite and other suchresins that have high tensile and impact strength over a broadtemperature range, are virtually shatter-proof and are extrudable astransparent sheets, over the handling system to prevent contamination ofthe OMDs and solutions as well as for the safety of the system operator.

2. Pre-coded memory devices

Alternatively, the matrices attached thereto may be encoded with apre-programmed identifying bar code, such as the 2-D optical bar codethat will be encoded on the matrix and read by laser. Such pre-codeddevices may be used in embodiments in which parameters, such as locationin an automated synthesizer, are monitored. The identity of a product orreactant determined by its location or path, which is monitored byreading the chip in each device and storing such information in a remotecomputer.

C. Data storage units with memory

In embodiments in which OMDs are used, the programmable devices areremote memories, such as computers into which information regarding theencoded information and linked molecules and biological particles isstored. The OMDs are read/write devices or are precoded devices.

For use with the matrices in which the memory, rather than a code, islinked to the device, any remotely programmable data storage device thatcan be linked to or used in proximity to the solid supports andmolecules and biological particles as described herein is intended foruse herein. Preferred devices are rapidly and readily programmable usingpenetrating electromagnetic radiation, such as radio frequency orvisible light lasers, operate with relatively low power, have fastaccess [preferably 1 sec or less, more preferably 10²-10³ sec], and areremotely programmable so that information can be stored or programmedand later retrieved from a distance, as permitted by the form of theelectromagnetic signal used for transmission. Presently preferreddevices are on the order of 1-10 mm in the largest dimension and areremotely programmable using RF, microwave frequencies or radar [see,e.g., Roland et al. (1996) Nature 381:120].

Recording devices may be active, which contain a power source, such as abattery, and passive, which does not include a power source. In apassive device, which has no independent power source, thetransmitter/receiver system, which transfers the data between therecording device and a host computer and which is preferably integratedon the same substrate as the memory, also supplies the power to programand retrieve the data stored in the memory. This is effected byintegrating a rectifier circuit onto the substrate to convert thereceived signal into an operating voltage.

Alternatively, an active device can include a battery [see, e.g., U.S.Pat. No. 5,442,940, U.S. Pat. No. 5,350,645, U.S. Pat. No. 5,212,315,U.S. Pat. No. 5,029,214, U.S. Pat. No. 4,960,983] to supply the power toprovide an operating voltage to the memory device. When a battery isused the memory can be an EEPROM, a DRAM, or other erasable memoryrequiring continuous power to retain information. It may be desirable tocombine the antenna/rectifier circuit combination with a battery tocreate a passive/active device, with the voltages supplied by eachsource supplementing each other. For example, the transmitted signalcould provide the voltage for writing and reading, while the battery, inaddition to supplementing this voltage, provides a refresh voltage for aDRAM memory so that data is retained when the transmitted signal isremoved.

The remotely programmable device can be programmed sequentially to beuniquely identifiable during and after stepwise synthesis ofmacromolecules or before, or during, or after selection of screenedmolecules. In certain embodiments herein, the data storage units areinformation carriers in which the functions of writing data and readingthe recorded data are empowered by an electromagnetic signal generatedand modulated by a remote host controller. Thus, the data storagedevices are inactive, except when exposed to the appropriateelectromagnetic signal. In an alternative embodiment, the devices may beoptically or magnetically programmable read/write devices.

1. Electromagnetically Programmable Devices

The programmable devices intended for use herein, include any devicethat can record or store data. A preferred device will be remotelyprogrammable and will be small, typically on the order of 10-20 mm³ [or10-20 mm in its largest dimension] or, preferably smaller. Any means forremote programming and data storage, including semiconductors andoptical storage media are intended for use herein. These include Yagiantennaes [see, e.g., Roland et al. (1996) Nature 381:120], diodes,magnetic tapes, and any other medium for storing information.

Also intended for use herein, are commercially available precodeddevices, such as identification and tracking devices for animals andmerchandise, such those used with and as security systems [see, e.g.,U.S. Pat. Nos. 4,652,528, 5,044,623, 5,099,226, 5,218,343, 5,323,704,4,333,072, 4,321,069, 4,318,658, 5,121,748, 5,214,409, 5,235,326,5,257,011 and 5,266,926], and devices used to tag animals. These devicesmay also be programmable using an RF signal. These device can bemodified, such as by folding it, to change geometry to render them moresuitable for use in the methods herein. Of particular interest hereinare devices sold by BioMedic Data Systems, Inc, NJ [see, e.g., theIPTT-100 purchased from BioMedic Data Systems, Inc., Maywood, N.J.; see,also U.S. Pat. Nos. 5,422, 636, 5,420,579, 5,262,772, 5,252,962,5,250,962, and see, also, U.S. application Ser. No. 08/322,644, filedOct. 13, 1994]. ID tags available from IDTAG™ Inc, particularly theIDT150 read/write transponder [ITDAG™ Ltd. Bracknell, Berks RG12 3XQ,UK, fabricated using standard procedures and the method for coilwinding, bonding and packaging described in International PCTapplication Nos. WO95/33246, WO95/16270, WO94/24642, WO93/12513,WO92/15105, WO91/16718; see, also U.S. Pat. Nos. 5,223,851, 5,261,615and 5,281,855] are also preferred herein. The IDT150 is a CMOS devicethat provides a kilobit of EEROM. This transponder also includes a 32bit fixed code serial number that uniquely identifies each chip. TheIDTAG™ transponder transmits data to a transceiver system by amplitudemodulating its coil and generating an EM field. It receives data andcommands from a transceiver by demodulating the field received by thecoil and decoding the commands. The transponder derives its power sourcefrom a frequency emitted in the signal from the reader, to which thetransponder emits a response. A smaller version [that has 16 bit EEROM]and is about 11 mm×4 mm×3 mm of this transponder is also among preferreddevices. These transponders are packaged in glass or polystyrene orother such material. Also preferred herein, are tags fabricated by andavailable from MIKRON under the name HITAG®[see, e.g., U.S. Pat. No.5,345,231 for a description of the systems for reading and writing].

In a preferred embodiment herein, the data storage unit includes asemi-conductor chip with integrated circuits formed thereon including amemory and its supporting circuitry. These devices can be written to andinterrogated from a distance. A radio frequency transmitter/receiversystem supplies power to program and retrieve data. In particular, thedata storage unit preferably includes a programmable read onlysemiconductor memory [PROM], preferably a non-volatile memory or othermemory that can store data for future retrieval, that will haveinformation describing or identifying the molecules or biologicalparticles linked to or in proximity to the matrix. This informationeither identifies the molecule or biological particles including a phageand viral particles, bacteria, cells and fragments thereof, provides ahistory of the synthesis of the molecule, or provides information, suchas a batch number, quality control data, reaction number, and/oridentity of the linked entity. The memory is programmed, before, duringor, preferably, after, each step of synthesis and can thereafter beread, thereby identifying the molecule or its components and order ofaddition, or process of synthesis.

While many well known read only memory devices use fuse structures thatare selectively “blown” to store data points, with a fuse located ateach possible data address in an array, among the devices of interestherein are those that rely on antifuse programming technology, in whichshort circuits are selectively created through an insulating layerseparating word and bit lines in an array. Due to the relatively lowlevel of voltage supplied by the transmitted signal when the memorydevice is passive, antifuse memories are readily used because of thelower voltage requirements for writing.

Thus, suitable memory devices, are about 1-20 mm in the smallestdimension [or smaller], are rapidly programmable [1 sec, preferably 1msec or less], can be interrogated from a distance [distances of about acentimenter up to about an inch are presently preferred], and areprogrammable using electro-magnetic radiation, preferably frequencies,such as those within the radio frequency range, that do not alter theassessed activities and physical properties of the molecules andbiological particles of interest.

Devices that rely on programmable volatile memories are also intendedfor use herein. For example, a battery may be used as to supply thepower to provide an operating voltage to the memory device. When abattery is used the memory can be an EEPROM, a DRAM, or other erasablememory requiring continuous power to retain information. It may beadvantageous to combine the antenna/rectifier circuitry with a batteryto create a passive/active device, in which the voltages supplied byeach source supplement each other. For example, the transmitted signalcould provide the voltage for writing and reading, while the battery, inaddition to supplementing this write/read voltage, provides a refreshvoltage for a DRAM memory so that data is retained when the transmittedsignal is removed. A 2 mm×2 mm×0.1 mm chip [or 3 mm×3 mm×0.1 mm] ispresently among the preferred chips [fabricated by Sokymat]. This chiphas monolithic antenna. Such chips addressable [read/write] usingmicrowave [Ghrtz] range frequencies.

a. Antifuses

An antifuse contains a layer of antifuse material sandwiched between twoconductive electrodes. The antifuse device is initially an opencircuited device in its unprogrammed state and can be irreversiblyconverted into an essentially short circuited device by the applicationof a programming voltage across the two electrodes to disrupt theantifuse material and create a low resistance current path between thetwo electrodes.

An exemplary antifuse structure for use herein is formed by defining aword line of heavily N-doped polysilicon on an insulating substrate,depositing an antifuse layer of lightly N-doped semiconductor over thepolysilicon, and defining a metal address [or bit] line upon and inelectrical contact with the antifuse layer. The semiconductor materialused for the antifuse layer is typically selected from among silicon,germanium, carbon and alpha-tin. The properties of the semiconductormaterial are such that the material is essentially non-conductive aslong as the voltage across it does not exceed a threshold level. Oncethe threshold voltage is exceeded, a conductive filament is formedthrough the semiconductor so that the resistance between the metal andpolysilicon lines at the points at which they cross irreversiblyswitches from a high resistance state to a relatively low resistancestate.

To program or change the resistance of the antifuse from a very highlevel [greater than 100,000,000 ohms] to a low level [less than 1000ohms], a voltage of sufficiently high electrical field strength isplaced across the antifuse film to create a short circuit. The voltagelevel required to induce breakdown is determined by the level of dopantin the antifuse layer. As breakdown occurs electrical current will flowthrough one small region of the film. The current is limited by theresistance of the filament itself as well as any series resistance ofconductive layers or logic devices [transistors] in series with theantifuse.

Examples of the antifuse and its use as a memory cell within a Read-OnlyMemory are discussed in Roesner et al., “Apparatus and Method of Use ofRadio frequency Identification Tags”, U.S. application Ser. No.08/379,923, filed Jan. 27, 1995, Roesner, “Method of Fabricating a HighDensity Programmable Read-Only Memory”, U.S. Pat. No. 4,796,074 (1989)and Roesner, “Electrically Programmable Read-Only Memory Stacked above aSemiconductor Substrate”, U.S. Pat. No. 4,442,507 (1984). A preferredantifuse is described in U.S. Pat. No. 5,095,362. “Method for reducingresistance for programmed antifuse” (1992) [see, also U.S. Pat. Nos.5,412,593 and 5,384,481].

U.S. Pat. No. 5,095,362 provides a method for fabricating a layer ofprogrammable material within an antifuse that exhibits relatively lowerthan normal resistance in its programmed state and also provides asemiconductor device containing an antifuse film of the type composed ofsemiconductor material having a first electrical state that ischaracterized by high electrical resistivity and a second electricalstate that is characterized by low electrical resistivity.

The means for selectively decreasing resistivity includes nonactivatedconductive dopants that are ion implanted within the otherwise highlyresistive semiconductor material. The dopants as implanted are in anonactivated state so that the dopants do not enhance the conduction ofcarriers in the film. Once activated, the dopants enhance the conductionof carriers in the film. Activation of the dopants occurs uponapplication of a threshold voltage across a predetermined and selectedportion of the material in which the dopants are disposed. The selectedportion is defined by the crossover point of selected word and bit [oraddress lines. The dopants are N-type, selected from among antimony,phosphorous, arsenic, and others to provide additional charge carriers.The implant dosage is used to determine the threshold voltage level thatwill be required to induce formation of the conductive filament. P-typedopants, such as boron, may also be used to affect a change inprogramming voltage.

b. A recording device with non-volatile memory

FIG. 5 depicts a recording device containing a non-volatileelectrically-programmable read-only memory (ROM) 56 that utilizesantifuse, EEPROM or other suitable memory, is combined on a singlesubstrate 54 with a thin-film planar antenna 60 forreceiving/transmitting an RF signal 58, a rectifier 62 for deriving avoltage from a received radio frequency (RF) signal, ananalog-to-digital converter (ADC) 64 for converting the voltage into adigital signal for storage of data in the memory, and adigital-to-analog converter (DAC) 66 for converting the digital datainto a voltage signal for transmission back to the host computer isprovided. A single substrate 54 is preferred to provide the smallestpossible chip, and to facilitate encapsulation of the chip with aprotective, polymer shell (or shell plus matrix or matrix material) 52.Shell 52 must be non-reactive with and impervious to the variousprocesses that the recording device is being used to track in order toassure the integrity of the memory device components on the chip.Materials for the shell include any such materials that are known tothose of skill in the art, including glasses, ceramics, plastics andother inert coatings.

Based on current semiconductor integrated circuit fabrication processcapabilities, in a preferred embodiment the finished chip on which allof the listed components are integrated is on the order of 1 mm×1 mm[˜40 mils×40 mils], with a memory capacity of about 1024 bits, but canhave greater or lesser capacity as required or desired. Greater memorycapacity, where needed, and smaller chips, however, will be preferred.

The chip may be larger to accommodate more memory if desired, or may besmaller as design rules permit smaller transistors and higher devicedensities, i.e., greater memory capacity.

The antifuse ROM structure described herein, and the method forfabricating the same, are based upon the known devices. (See, e.g., U.S.Pat. No. 4,424,579, No. 4,442,507, No. 4,796,074, No. 5,095,362, No.4,598,386, No. 5,148,256, No. 5,296,722, and No. 5,583,819.)

In an antifuse-type memory device, the individual memory cells arearranged in arrays of orthogonal conductive word and bit lines to obtainthe smallest possible memory array size. For example, for 1024 bits ofmemory, there are 32 word lines and 32 bit lines for a square array.Memories with greater capacity may also be used. Schottky diodes areformed generally corresponding to the points at which the word and bitlines cross. The word and bit lines are separated by an undoped orlightly-doped semiconductor layer with interstitial doping. Thesemiconductor layer may also be amorphous silicon with implanted dopantsin a nonactivated state. Each of these crossover points is a memory celland is the equivalent of a programmable switch in series with a Schottkydiode. Data are stored by the switch being ON or OFF. As fabricated, anantifuse memory device has all of its switches in the OFF state. Aswitch is turned on by applying a voltage in excess of a pre-determinedthreshold voltage to one of the word lines while setting a selected bitline to a low logic level. The threshold voltage is determined by theimpedance of the semiconductor layer, i.e., its doping level. Accordingto the process for fabricating the antifuse memory of the preferredembodiment, the impedance can be less than 200 ohms with a thresholdvoltage for programming as low as 3 volts. Since in the embodimentdescribed herein the programming voltage is provided solely by therectified RF signal, a low threshold is preferred. Application ofvoltage exceeding the threshold activates the interstitial dopant in thesemiconducting film at the point corresponding to the cross-over betweenthe two lines, causing a short between the word and bit lines andirreversibly turning on that particular switch or memory cell. Addressdecoders, as are known in the art, are used to selectively address theword and bit lines for purposes of writing information to and readingstored information from the memory array. [See, e.g., U.S. Pat. No.5,033,623, 5,099,226, 5,105,190, 5,218,343, 5,323,7041. Exemplary meansfor decoding information to be stored in memory and to be read frommemory are provided in U.S. Pat. Nos. 4,442,507 and No. 4,598,386.

Information to be written into the memory need not be detailed since thedata stored in the memory is primarily acting as an identificationmarker that is traceable to a more detailed record stored in the hostcomputer memory 68, independent of the memory associated with the matrixsupport or tagged molecule or biological particle. In this manner, theRF signal from transmitter 50 that is used to provide the power and thesignal to the matrix particle memory need only address a single memorycell to indicate that a nascent oligomer linked to or in proximity tothe memory device has been subjected to a given process step or toidentify a molecule or biological particle. Thus, a sophisticated memoryaddressing system need not be provided on the matrix particle memorychip, and shift registers may be used to control memory addressing.Alternatively, a microprocessor which is mask-programmed during thefabrication process for controlling an address bus which connects theADC 64 and the DAC 66 to the memory array may also be built onto thesame substrate on which the memory and other components are integrated.Other integrated means for selectively addressing locations within thememory are known and will be apparent to the practitioner skilled in theart.

As described above, antifuse memories are well known in the art. Thesememories include structures in which the word and bit lines may be madeof either N+polysilicon or metal [aluminum or aluminum-silicon],separated by silicon dioxide (SiO₂), silicon nitride (Si₃N₄),combinations thereof, or amorphous silicon alone or in combination withSiO₂ and/or Si₃N₄. In each case, a short circuit is created at locationsin the antifuse material corresponding to the crossover location ofselected word and bit lines by applying a voltage in excess of apre-determined threshold voltage.

Examples of alternate means for forming an antifuse memory are providedin the following U.S. Pat. No. 5,248,632, issued Sep. 28, 1993, of Tunget al.; U.S. Pat. No. 5,250,459, issued Oct. 5, 1993, of Lee, No.5,282,158, issued Jan. 25, 1994, of Lee; No. 5,290,734, issued Mar. 1,1994, of Boardman, et al.; U.S. Pat. No. 5,300,456, issued Apr. 5, 1994,of Tigelaar et al.; U.S. Pat. No. 5,311,039, issued May 10, 1994, ofKimura, et al.; U.S. Pat. No. 5,316,971, issued May 31, 1994, of Chianget al.; U.S. Pat. No. 5,322,812, issued Jun. 21, 1994, of Dixit, et al.;U.S. Pat. No. 5,334,880, issued Aug. 2, 1994, of Abadeer, et al., andothers.

Generally for use in the methods herein, non-volatility of the memory orthe ability to lock or prevent erasure is preferred since power isapplied to the chip only when it is subjected to the RF or othertransmission signal for reading or reading and writing. Furtherconsiderations are the voltage levels required for writing into memory,since the threshold voltage must be less than the maximum voltage of therectified RF signal in order to assure that sufficient voltage is alwaysavailable during the writing process. The write voltage may be enhancedby supplementing the RF-supplied voltage with optically-generatedvoltage, such as a photocell. Photocells on semiconductor substrates arewell known in the art and could be easily integrated onto the chip. Alaser or other light source could be readily included in the writeapparatus to illuminate the chip coincident with transmission of the RFwrite signal. Similarly, other forms of electromagnetic radiation may beused to provide additional power, if needed.

Although antifuse memories are not designed to be erasable, it may bedesirable to re-use the devices if the memory becomes full. In suchinstances, conventional electrically programmable erasable read onlymemories [EEPROMs] may be used instead. Since EEPROMs require higherwrite voltage levels, it may be desirable to supplement the RF-suppliedvoltage as described above. In EEPROMs, stored data can be erased byexposing the device to UV light.

Signal rectifier 62 may be one or more Schottky diode(s), making itreadily incorporated into the fabrication process used for the memoryarray. Other means for signal rectification may be used as are known.The ADC 64 and DAC 66 are well-known devices and are readily integratedonto the substrate 54 using the fabrication process described in thereferences for the memory array. Radio frequency modulation techniques,which are known in the art, for example, pulse code modulation, may beadapted to permit direct digital transmission, in which case the ADC andDAC may not be required.

Antenna 60 is formed during the fabrication process using conventionalphotolithographic techniques to provide one or more metal structures,such as aluminum, to receive a pre-determined wavelength RFtransmission. The antenna may be a simple straight line half-waveantenna which is created by patterning a structure during the secondmetal process steps so that the structure has a length equal to one-halfof the wavelength of the selected RF transmission frequency in freespace. Another option for formation of the antenna is as a small loop,either on a dedicated portion of the chip, or encircling the othercomponents of the chip, also formed during the second metal step of thefabrication process. It is noted that, in a typical semiconductorfabrication process, such as would be compatible with the preferredantifuse memory, the first and second metal steps include depositing alayer of aluminum, then patterning the aluminum photolithographicallyfollowed by a plasma etch to define the desired features. Except wherevias are formed, the two metal layers are separated by a dielectricfilm. Dipole antennas may be formed by patterning the second metal in asimilar manner, with the dimensions of the antenna being selected forthe appropriate RF frequency. The two metal layers may also be used toform a microstrip antenna structure by selecting the dielectric filmbetween the metal layers such that it has a dielectric constant andthickness appropriate so that the microstrip is resonant at one-half ofthe RF wavelength. (The first metal layer provides the ground plane.)The metal structures, which may be square patches, circles, lines, orother geometries, are defined photolithographically during the normalmasking steps of the first and second metal processes. Other antennastructures which can be configured as a thin film device for integrationonto a common substrate with the memory structure and other componentsmay be used and will be apparent to those skilled in the art. Similarly,a resonant circuit (inductor-capacitor) can be readily integrated ontothe chip, with the resonant circuit being tuned to the RF carrier signalof the transmitter.

Frequency tuning of either an antenna or resonant circuit can provideadditional coding capability. For example, a first group of memorydevices can be tuned to receive a carrier wave of a first RF frequency,e.g., f₁, and a second group could be tuned to receive a secondfrequency f₂, and so on. The separate carrier frequencies could provideadditional means for tracking or providing information to the devices,even if the groups become intermixed.

The RF antenna may, in an alternate embodiment, be formed external tothe semiconductor substrate. In this configuration, a separateconductive wire, which acts as an antenna, will be attached to a bondpad formed on the chip using methods known to those skilled in the art.The wire will then be stabilized when the chip is encased in theprotective shell, so that the antenna extends at some angle to the chip.

Also, as an alternative to signal transmission via RF, the antifuse orother semiconductor memory and supporting circuitry can receive theaddressing commands and device power by optical transmission. In thisembodiment, the RF antenna 60 would be replaced by a photocell thatgenerates sufficient write voltage to exceed the threshold voltage. Forthe addressing commands, the RF transmitter 50 is replaced by a lightsource, and the commands may be transmitted digitally by pulsing theoptical transmitter, which can be a laser, flash lamp or other highintensity light source. It is noted that the light intensity must besufficient to generate adequate voltage, either singly or in conjunctionwith a second power generating device, in the photocell to write intomemory, but not so high that it damages the metal interconnect on thechip. With digital data transmission analog-to-digital anddigital-to-analog conversion circuitry can be eliminated.

c. Other Memory Devices

Other types of electrically-programmable read-only memories, preferablynon-volatile memories, which are known in the art, may be used [see,e.g., U.S. Pat. No. 5,335,219]. Chips, such as those sold by Actel,Mosaic, Lattice Semiconductor, AVID, Anicare, Destron, Rayethon, Altera,ICT, Xilinix, Intel and Signetics [see, e.g., U.S. Pat. Nos. 4,652,528,5,044,623, 5,099,226, 5,218,343, 5,323,704, 4,333,072, 4,321,069,4,318,658, 5,121,748, 5,214,409, 5,235,326, 5,257,011 and 5,266,926] maybe used herein. Preprogrammed remotely addressable identification tags,such as those used for tracking objects or animals [see, e.g., U.S. Pat.Nos. 5,257,011, 5,235,326, 5,226,926, 5,214,409, 4,333,072, availablefrom AVID, Norco, Calif.; see, also U.S. Pat. Nos. 5,218,189, 5,416,486,4,952,928, 5,359,250] and remotely writable versions thereof are alsocontemplated for use herein. Preprogrammed tags may be used inembodiments, such as those in which tracking of linked molecules isdesired. Devices sold by XCI [San Jose, Calif.] that operate in thelower frequency [˜900 mhz] range are also preferred herein.

d. Pre-coded Memory Devices

Alternatively, the matrices or strips attached thereto may be encodedwith a pre-programmed identifying bar code, such as an optical bar codethat will be encoded on the matrix and read by laser. Such pre-codeddevices may be used in embodiments in which parameters, such as locationin an automated synthesizer, are monitored. The identity of a product orreactant determined by its location or path, which is monitored byreading the chip in each device and storing such information in a remotecomputer. Read/write tags such as the IPTT-100 [BioMedic Data Systems,Inc., Maywood, N.J.; see, also U.S. Pat. Nos. 5,422,636, 5,420,579,5,262,772, 5,252,962, 5,250,962, and U.S. application Ser. No.08/322,644] are also contemplated for use herein.

Among the particularly preferred devices are the chips [particularly,the IPTT-100, Bio Medic Data Systems, Inc., Maywood, N.J.; see, alsoU.S. Pat. Nos. 5,422,636, 5,420,579, 5,262,772, 5,252,962 and 5,250,962and U.S. application Ser. No. 08/322,644,] that can be remotely encodedand remotely read. These devices, such as the IPTT-100 transponders thatare about 8 mm long, include a recording device, an EEPROM, a passivetransponder for receiving an input signal and transmitting an outputsignal in response. In some embodiments here, the devices are modifiedfor use herein by altering the geometry. They are folded in half and theantenna wrapped around the resulting folded structure. This permitsconvenient insertion into the microvessels and formation of othercombinations.

These devices include a power antenna means [see, e.g., U.S. Pat. No.5,250,944 and U.S. Pat. No. 5,420,579] for receiving the input signal,frequency generator and modulator means for receiving the input signalthe receive antenna means and for generating the output signal. Theoutput signal has a frequency different from the input frequency,outputs the output signal in response the input signal. The input signalhaving a first frequency, the output signal has a second frequency thatis a multiple of the first frequency, and is greater that the firstfrequency. It also includes a transmitting antenna means for receivingthe output signal from the frequency generator and modulator means andthat transmit the output signal. Data are stored within the transponderwithin a reprogrammable memory circuit that is programmed by the user[see, e.g., U.S. Pat. No. 5,422,636 and EP 0 526 173 A3]. A transponderscanner for scanning and programming the transponder is also available[Bio Medic Data Systems Inc. DAS-5001 CONSOLE™ System, e.g., U.S. Pat.No. 5,252,962 and U.S. Pat. No. 5,262,772].

e. Other Memories

Another such device is a 4 mm chip with an onboard antenna and an EEPROM[Dimensional Technology International, Germany]. This device can also bewritten to and read from remotely.

ID tags available from IDTAG™ Inc, particularly the IDT150 read/writetransponder [ITDAG™ Ltd. Bracknell, Berks RG12 3XQ, UK], discussedabove, are also preferred herein.

f. Monolithic Semiconductors

Additionally, smaller [about 2 mm×2 mm×0.1 mm or less] monolithicdevices are of interest herein. For example, in a particular embodimentof an electromagnetically programmable tag, such as, for exemplificationpurposes an RF tag, a single chip tag is formed entirely on a singlesubstrate. More specifically, referring to FIG. 51, a monolithic tag,such as an RF tag, is shown and generally designated 4700. Thismonolithic tag is sized such that the substrate has following overallexternal dimensions: width 4710 of 2 mm wide, length 4708 of 2 mm, and aheight 4714 of 0.1 mm. As a result of this miniaturization of the tag, avariety of shapes and sizes of the tags may be created [see, e.g., U.S.Pat. No. 4,857,893 issued to Carroll in 1989, entitled “Single ChipTransponder Device”, which describes a single substrate (monolithic) RFtransponder which, due to its single substrate, is simple tomanufacture].

In FIG. 51, the tag 4700 is shown having a substrate 4702 is formed withan antenna 4714. This antenna is preferably formed on the substrateusing a metalization process wherein a metal is placed on a pattern onthe top surface of the substrate to create a particular antenna. Asshown, the antenna is substantially square, tracing out a coiled antennabeginning at pad 4712, and ending at conductor 4706 which attaches backto the circuitry 4704. It should be appreciated that while the antennais shown to be square, any other shapes could be used. In particular, acircular antenna could be formed just as easily on the surface of thesubstrate. It should be appreciated, however, that the functionality ofthe antenna are likely very similar between a square antenna and acircular antenna. In addition to the antenna as shown, there may be asecond antenna on the back side of the substrate (not shown) which couldbe used to increase the number of windings or, as an alternative, betuned for a different frequency range than the antenna patterned on theupper surface of the substrate 4702.

Referring to FIG. 52, the tag is shown in plan view and has circuitry4704 which includes specific logic and control electronics generallydenoted 4706. The pattern of the antenna 4714 is easily appreciated fromthis view. Moreover, the circular equivalent can be easily envisioned onthe substrate 4702 to spiral around the circuitry 4704 to create asimilarly sized antenna.

The specific electronic circuitry that is contained in circuitry 4704 isknown and well described [see, e.g., U.S. Pat. No. 4,857,893, and U.S.Pat. No. 5,345,231, discussed above] For example, U.S. Pat. No.5,345,231, entitled “Contactless Inductive Data-Transmission System”discloses circuitry capable of communicating identification informationacross a wireless communication system which employs an inductivecoupling. Specifically, with reference to FIG. 2 of that reference, thisinductive coupling provides the power to run the tag electronics, aswell as provides the communication channel with which the identificationinformation travels. The circuitry includes a rectifier attached to theantenna to receive an electromagnetically coupled signal, and to createits own power from the signal. In addition to the rectifier, a clockextractor and demodulator also receive the antenna's signal. The clockextractor recreates a communication clock, and the demodulator decodesthe signal received from the antenna using that clocking information.This information is provided to a control unit which either programs ordownloads the contents of a memory bank. In the particular tag discussedherein, the memory bank can include a single data bit, or may be easilyexpanded as is generally known in the art to a variety of memory sizes,up to several kilobytes. Once the memory has been accessed, the controlunit can communicate with the base transmitter/receiver by sending datato the modulator which is also electrically connected to the antenna. Inthat manner, the antenna can be used to either receive a signal from thetransmitter, or to transmit a signal to the receiver.

The antenna for use with these particular electronics is tuned for aresonant frequency of approximately 125 kHz. It is to be appreciated, asdiscussed above, that the antenna can easily be tuned to receive avariety of frequencies, including 125 kHz. It should be noted, however,that depending on the frequencies to be received, the antenna shape andsize could be altered. In fact, it should be appreciated that a singleantenna which is capable of receiving a number of frequencies could becreated by having electrical leads which attached to a number of pointsalong the length of the antenna. As a result, a single antenna could beused to receive a number of frequencies, where the frequency to bereceived could be set by an initial communication with the tag. Becauseof such frequency specification, it should be appreciated that the anumber of tags could be addressed simultaneously in a “batch” read orwrite process.

A batch read/write process enables a single transmitter station toaddress more than one tag at the same time. This is particularly usefulwhen desiring to program a number of tags with the same or similarinformation. Thus, by batch writing the information to a number of tags,the programming process is shortened and simplified, while at the sametime, minimizing the opportunity for error.

In addition to the ease and accuracy of batch accessing the tags, such afeature is particularly useful when faced with a large number of tags toidentify. More particularly, the ability to batch access the tagspermits the transmitter/receiver to identify any number of tags withinan area simply by one access process instead of having to access eachtag individually. It should be appreciated that in addition to simplyaccessing all tags at one time, there could be a variety of signal typeswhich could narrow the field of access. One manner of restricting theaccess to tags, such as an RF tag, could be to make any interrogationeither code or frequency discriminating. Such discrimination wouldoccur, for example, by selecting only the particular RF tags within afrequency range. On the other hand, the specific address code of anumber of RF tags could be programmed such that by identifying, forexample, the first four of an eight bit address scheme, only a portionof the RF tags identifiable with those eight addressing bits could beaddressed.

The circuitry for communication with a transmitting and receiving baseis known [see, e.g., U.S. Pat. No. 4,857,893, entitled “Single ChipTransponder Device”, see esp. FIG. 2 of that reference]. Briefly, anantenna receives a carrier signal which is provided to a rectifier anddemodulator, as well as a timing decoder. The rectifier captures aportion of the carrier signal to derive dc power to drive the RF tagitself. The timing decoder uses the remaining portion of the antennasignal to derive the timing signals necessary to control the datastorage and generation of the RF tag. Once accessed, the data generatorcreates a data signal which is modulated and supplied to the antenna forretransmit back to the transmitter/receiver unit.

Referring now to FIG. 53, the single substrate tag shown in FIGS. 51 and52 is shown attached to a stirring bar, which is commonly known in theindustry. The stirring bar includes a material which is capable ofmagnetic interaction. This material is then encapsulated in an inertmaterial such as a polymer which insulates the material from theenvironment. As shown partially cut away for clarity, the insulationmaterial 4718 covers the entire outer surface of the material 4716.Prior to encapsulation, the monolithic RF tag 4700 is attached to thematerial such that, once encapsulated, the RF tag is also encapsulated.

Once a stirring bar is equipped with an RF tag, the stirring bar may beplaced in a container and the container may then be easily trackedthrough any environment simply by placing the container over anidentification station. Moreover, by placing a number of stirring barsin different containers, with each stirring bar having its ownidentification number, virtually an unlimited number of containers maybe tracked. This would be particularly useful in environments wherethere is a need to manipulate a large number of containers, such as abiomedical laboratory. It should be appreciated, however, that inaddition to placing the monolithic RF tag on a stirring bar, the tag maybe attached to any other commonly used devices to facilitate trackingthose devices. For instance, as described elsewhere herein, eachcontainer may be manufactured with the monolithic tags embedded in thecontainer or in sleeve that is removable attached to a container. Thispermits tracking of the container throughout its environment, withoutthe need to add any device or item to the contents of the container.

In addition to allowing the simple tracking of various containers, it ispossible to place a plurality of tags, such as different RF tags, withina container that is already tagged. More specifically, it is possible toplace a tag in a container that is already identified with one tag. Thiscombining of multiple tags would enable a greater level of tracking ofthe container. For instance, a beaker could be formed with a RF tagintegral to its structure. Once identified, the beaker may be filledwith a variety of materials, each having its own identification number.Thus, when solution B is added to beaker A,. an RF tag indicating thematerial B can simply be dropped into the beaker A. Likewise, whensolution C is added to beaker A, an RF tag indicating the material C canbe dropped in the beaker. As a result of this marking method, it wouldbe possible to verify the exact contents of a container. Specifically,by reading the various RF tags within the beaker A (solution B andsolution C) the entire contents of the container would be identified.

Alternatively, in addition to, or instead of, simply identifying thecontents of a container, it would also be possible to track thewhereabouts of a container by adding identifying RF tags at variouslocations in its path. For instance, a beaker could be marked with anidentifying RF tag A, and an identifying RF tag B could be added when aparticular process is performed on the contents of the beaker A.Similarly, an identifying RF tag C could be added to the beaker at thenext process step. Thus, the RF tags in the beaker would indicate theexact historical location of the container A simply by decoding thecontents of the RF tags contained therein.

In addition, the RF tags could be used to provide a combination of theabove described contents and location based information. Thiscombination would provide a means to analyze a “chain of possession” forvarious contents of a container. In other words, the contents of abeaker could be determined by identifying the RF tags. This informationwould provide a history of the contents of the container, as well aslocation of the processes which were performed on the container.

2. Optically or Magnetically Programmed Devices

In addition to electrically-programmable means for storing informationon the matrix particles, and the 2-D bar codes, described above, otheroptical and magnetic means may be used. Such optical storage means areknown [see, e.g., U.S. Pat. No. 5,136,572, issued Aug. 4, 1992, toBradley]. Here, an array of stabilized diode lasers emits fixedwavelengths, each laser emitting light at a different wavelength.Alternatively, a tunable diode laser or a tunable dye laser, each ofwhich is capable of emitting light across a relatively wide band ofwavelengths, may be used. The recording medium is photochemically activeso that exposure to laser light of the appropriate wavelength will formspectral holes.

As illustrated In FIG. 8, an optical write/read system is configuredsimilar to that of the embodiment of FIG. 7, with a vessel 164containing a number of the particles which are separated and oriented bypassing through a constricted outlet into a write/read path 158 that hasan optically-transparent tube (i.e., optically transparent to therequired wavelength(s)) with a cross-section which orients the particlesas required to expose the memory surface to the laser 152 which iscapable of emitting a plurality of discrete, stable wavelengths. Gatingand detection similar to that described for the previous embodiment maybe used and are not shown. Computer 154 controls the tuning of laser 152so that it emits light at a unique wavelength to record a data point.Memory within computer 154 stores a record indicating which process stepcorresponds to which wavelength. For example, for process A, wavelengthλ 1 , e.g., 630 nm (red), for process C, λ 2 , e.g., 550 nm (yellow),and for process E, λ 3 , e.g., 480 nm (blue), etc. The recording medium156 is configured to permit orientation to repeatably expose therecording side of the medium to the laser beam each time it passesthrough tube 158. One possible configuration, as illustrated here, is adisc.

To write onto the recording medium 156, the laser 152 emits light of theselected wavelength to form a spectral hole in the medium. The light isfocused by lens 160 to illuminate a spot on recording medium 156. Thelaser power must be sufficient to form the spectral hole. For reading,the same wavelength is selected at a lower power. Only this wavelengthwill pass through the spectral hole, where it is detected by detector162, which provides a signal to computer 154 indicative of the recordedwavelength. Because different wavelengths are used, multiple spectralholes can be superimposed so that the recording medium can be very smallfor purposes of tagging. To provide an analogy to the electrical memoryembodiments, each different wavelength of light corresponds to anaddress, so that each laser writes one bit of data. If a large number ofdifferent steps are to be performed for which each requires a uniquedata point, the recording media will need to be sufficiently sensitive,and the lasers well-stability, to vary one within a narrow band toassure that each bit recorded in the media is distinguishable. Sinceonly a single bit of information is required to tag the particle at anygiven step, the creation of a single spectral hole at a specificwavelength is capable of providing all of the information needed. Thehost computer then makes a record associating the process performed witha particular laser wavelength.

For reading, the same wavelength laser that was used to create thespectral hole will be the only light transmitted through the hole. Sincethe spectral holes cannot be altered except by a laser having sufficientpower to create additional holes, this type of memory is effectivelynon-volatile. Further, the recording medium itself does not have anyoperations occurring within its structure, as is the case in electricalmemories, so its structure is quite simple. Since the recording mediumis photochemically active, it must be well encased within an opticallytransmissive [to the active optical wavelength(s)], inert material toprevent reaction with the various processing substances while stillpermitting the laser light to impinge upon the medium. In many cases,the photochemical recording media may be erased by exposure to broadspectrum light, allowing the memory to be reused.

Writing techniques can also include the formation of pits in the medium.To read these pits, the detector 162 with be positioned on the same sideof the write/read tube 158 as the laser 152 to detect light reflectedback from the medium. Other types of optical data storage and recordingmedia may be used as are known in the art. For example, optical discs,which are typically plastic-encapsulated metals, such as aluminum, maybe miniaturized, and written to and read from using conventional opticaldisc technology. In such a system, the miniature discs must be alignedin a planar fashion to permit writing and reading. Other opticalrecording media that may be appropriate for use in the recording devicesand combinations herein include, but are not limited to, magneto-opticalmaterials, which provide the advantage of erasability, photochromicmaterials, photoferroelectric materials, photoconductive electro-opticmaterials, all of which utilize polarized light for writing and/orreading, as is known in the art. When using any form of opticalrecording, however, considerations must be made to insure that theselected wavelength of light will not affect or interfere with reactionsof the molecules or biological particles linked to or in proximity tomatrix particles.

a. Three Dimensional Optical Memories

3-D memory storage devices include persistent hole burning, phaseholograms, and two photon optical 3-D memories that use organicmaterials and biomolecules. Any such devices are intended for useherein. Of particular interest are those that use organic materials andbiomolecules. Such memories can be incorporated into the matrixmaterials or inert polymeric materials that are derivatized for use asmatrices.

b. 3-D Optical Memories and Apparatus Therefor

Optical memory systems are based on light-induced changes in the opticalchemical or physical properties of materials. As such these memories areideally suited for use in the methods herein and in combination withmatrices, since the materials that form the memory may be incorporatedinto or part of the material from which the matrix is fabricated.

Polymer-based photonic materials that can store 1 trillion bytes of dateper cc have been developed [see, e.g., U.S. Pat. Nos. 5,268,862,5,130,362, 5,325,324; see, also, Dvornikov et al. (1996) Opt. Commun.128:205-210; Dvornikov et al. (1996) Res. Chem. Intermed. 22:115-28;Dvornikov et al. (1994) Proc. SPIE-Int. Soc. Ort. Eng. 2297:447-51;Dvornikov et al. (1994) Mol. Cryst. Liq. Cryst. Sci. Technol., Sect. A246:379-88; Dvornikov et al. (1994) J. Phys. Chem. 98:6746-52; Ford etal. (1993) Proc. SPIE-Int. Soc. Opt. 2026:604-613; Ford et al. Proc.SPIE-Int. Soc. Ort. Eng. 1853:5-13; Malkin et al. Res. Chem. Intermed.19:159-89; Dvornikov et al. (1993) Proc. SPIE-Int. Soc. Opt. Eng.1852:243-52; Dvornikov et al. (1992) Proc. SPIE-Int. Soc. Opt. Eng.1662:197-204; Prasad et al. (1996) Mater. Res. Soc. Symp. Proc.413:203-213; and Dagani in Chemical and Eng. News Sep. 23, 1996, pp.68-69]. This technology involves using a laser to encode information ina polymeric medium containing dye molecules that have a nonlinearoptical property known to those of skill in the art as two-photonabsorption. When the dye molecule is irradiated with light ofsufficiently high intensity i, it absorbs two photons of lightsimultaneously; the molecule then emits a photon of higher energy. Thismeans that the material can be irradiated with lower energy penetratinglight, such infrared or near infrared and produce a higher energyemission in the visible.

In these methods, the writing beam “photobleaches” spots in therecording medium so that those spots when subsequently illuminated witha reading beam will emit either no light or less light than thesurrounding medium [see, e.g., U.S. Pat. No. 5,325,324; see, also U.S.Pat. No. 5,130,362]. By varying the intensity of the writing laser, theextent of photobleaching can be varied to get a gray-scale, therebypermitting storage of information in analog as well as digital form.Dyes have been developed that are particularly suitable for use in thesemethods. For example, a stilbene derivative with substituted amino andsulfonyl groups [APSS] has been developed that has very strong 2-photonabsorption. The dye is dispersed in a polymer, such as a methacrylatepolymer, which then serves as a read/write medium.

Memories based on photochromaic materials, such as1-nitro-2-naphthaldehyde and the colorless base form of the laser dyerhodamine B, are also available [see, e.g., Dvornikov et al. (1996) Res.Chem. Intermed. 22:115-28].

As noted above, incorporation of the these dyes or other such moleculesinto the polymeric supports used in the syntheses and assays describedherein will permit the supports [or portions thereof] to serve asmemories to which information can be written and from which it can beread. Thus, for example, instead of using a symbology as described inthe embodiments herein, the polymer from which support is made willcontain a dye molecule or other molecule that exhibits 2-photonabsorption, and thereby serve as a storage medium that can be read orcan be a read/write medium. The other portion of the device can beradiation grafted and used as a support for chemical syntheses andassays.

c. Rhodopsins

Another memory means that is suitable for use in the matrix with memorycombinations are optical memories that employ rhodopsins, particularlybacteriorhodopsin [BR], or other photochromic substances that changebetween two light absorbing states in response to light of each of twowavelengths [see, e.g., U.S. Pat. Nos. 5,346,789, 5,253,198 and5,228,001; see, also Birge (1990) Ann. Rev. Phys. Chem 41:683-733].These substances, particularly BR, exhibit useful photochromic andoptoelectrical properties. BR, for example, has extremely large opticalnonlinearities, and is capable of producing photoinduced electricalsignals whose polarity depends on the prior exposure of the material tolight of various wavelengths as well as on the wavelength of the lightused to induce the signal. There properties are useful for informationstorage and computation. Numerous applications of this material havebeen designed, including its use as an ultrafast photosignal detector,its use for dynamic holographic recording, and its use for data storage,which is of interest herein.

The rhodopsins include the visual rhodopsins, which are responsible forthe conversion of light into nerve impulses in the image resolving eyesof mollusks, anthropods, and vertebrates, and also bacteriorhodopsin[BR]. These proteins also include a class of proteins that servephotosynthetic and phototactic functions. The best known BR is the onlyprotein found in nature in a crystalline membrane, called the “purplemembrane” of Halobacterium Halobium. This membrane converts light intoenergy via photon-activated transmembrane proton pumping. Upon theabsorption of light, the BR molecule undergoes several structuraltransformations in a well-defined photocycle in which energy is storedin a proton gradient formed upon absorption of light energy. This protongradient is subsequently utilized to synthesize energy-rich ATP.

The structural changes that occur in the process of light-induced protonpumping of BR are reflected in alterations of the absorption spectra ofthe molecule. These changes are cyclic, and under usual physiologicalconditions bring the molecule back to its initial BR state after theabsorption of light in about 10 milliseconds. In less than a picosecondafter BR absorbs a photon, the BR produces an intermediate, known as the“J” state, which has a red-shifted absorption maximum. This is the onlylight-driven event in the photocycle; the rest of the steps arethermally driven processes that occur naturally. The first form, orstate, following the photon-induced step is called “K”, which representsthe first form of light-activated BR that can be stabilized by reducingthe temperature to 90° K. This form occurs about 3 picoseconds after theJ intermediate at room temperature. Two microseconds later there occursan “L” intermediate state which is, in turn, followed in 50 microsecondsby an “M” intermediate state.

There are two important properties associated with all of theintermediate states of this material. The first is their ability to bephotochemically converted back to the basic BR state. Under conditionswhere a particular intermediate is made stable, illumination with lightat a wavelength corresponding to the absorption of the intermediatestate in question results in regeneration of the BR state. In addition,the BR state and intermediates exhibit large two-photon absorptionprocesses which can be used to induce interconversions among differentstates.

The second important property is light-induced vectorial chargetransport within the molecule. In an oriented BR film, such a chargetransport can be detected as an electric signal. The electrical polarityof the signal depends on the physical orientation of molecules withinthe material as well as on the photochemical reaction induced. Thelatter effect is due to the dependence of charge transport direction onwhich intermediates [including the BR state] are involved in thephotochemical reaction of interest. For example, the polarity of anelectrical signal associated with one BR photochemical reaction isopposite to that associated with a second BR photochemical reaction. Thelatter reaction can be induced by light with a wavelength around 412 nmand is completed in 200 ns.

In addition to the large quantum yields and distinct absorptions of BRand M, the BR molecule [and purple membrane] has several intrinsicproperties of importance in optics. First, this molecule exhibits alarge two-photon absorption cross section. Second, the crystallinenature and adaptation to high salt environments makes the purplemembrane very resistant to degeneration by environmental perturbationsand thus, unlike other biological materials, it does not require specialstorage. Dry films of purple membrane have been stored for several yearswithout degradation. Furthermore, the molecule is very resistant tophotochemical degradation.

Thus, numerous optical devices, including recording devices have beendesigned that use BR or other rhodopsin as the recording medium [see,e.g., U.S. Pat. Nos. 5,346,789, 5,253,198 and 5,228,001; see, also Birge(1990) Ann. Rev. Phys. Chem 41:683-733]. Such recording devices may beemployed in the methods and combinations provided herein.

3. Event-detecting Embodiment and Sensors

Combinations of the matrices with memories and sensors, such asbiosensors and devices that measure external parameters are provided.Combinations of memories with sensors are also provided. Variousembodiments of the sensors are set forth in the Examples. See, also FIG.9 and the description below. Examples of glucose sensors, calciumsensors, urea sensors, and intracranial pressure monitors are providedherein. In each embodiment, a memory is included as a means to trackpatient history and/or to store or record sensed information.

In particular, the memories and memories with matrices provided hereinmay be advantageously used in combination with sensors, which aredevices that measure external parameters, such as pH, temperature, ionconcentrations in solutions, and also biosensors, particularlyimplantable biosensors, which are used to measure internal parameters,such as electrolytes, blood glucose, to monitor blood pressure, andintracranial pressure.

The memories will be combined with sensors known to those of skill inthe art as described herein [see, Examples], and used to track thedevices, to detect reactions or event, to detect and store the detectedinformation, and to permit remote monitoring of patients or samples.

Also provided herein, are embodiments of matrices with memories in whichthe matrix is a sol-gel. The sol-gels, which are used to encapsulatebiological molecules, such as molecules used as sensors, will include amemory device. For example, sol-gel biosensors are known. Theimprovement herein provides a means to store information regarding thesensed event or detect the event.

Also provided herein, are implantable biosensors that are coated with anangiogenic agent, such as an FGF, a VEGF, a PDGF, IL-8 and others. Uponimplantation of the biosensor, agents will aid in directly blood flow tothe device, thereby permitting the sensor to detect internal conditionsmore accurately and for an extended period of time.

The biosensors may also be combined with conducting polymers [see,Barisci et al. (1996) “Conducting Polymer Sensors”, TRIP 4:307-311 toproduce, for example, implantable drug delivery devices. These materialsare polymers into which physiologically active substances, such as ahormone or enzyme, can be incorporated during or after copolymerization,and which will release the physiologically active substance uponapplication of a voltage. Combination of such polymers with amicroprocessor with memory will permit timed release of the activemolecule by the microprocessor. A second electrode 6510 coated with anelectroactive polymer 6512 containing appropriate chemical dye is usedto release the chemical dye for marking the bound antibodies andantigens.

a. Event-detecting Sensor

Another embodiment of the combinations herein uses a recording devicethat can detect the occurrence of a reaction or event or the status ofany external parameter, such as pH or temperature, and record suchoccurrence or parameter in the memory, such embodiment is hereinreferred to as a sensor. Any of the above-described matrices withmemories or memories may be modified to permit such detection. Forexample, the chip with the memory array with decoder, rectifiercomponents and antenna, such as RF antenna, can be modified by additionof a photodetector and accompanying amplifier components as shown inFIG. 9. The photodetector will be selected so that it is sensitive tothe frequencies of expected photoemissions from reactions of interest.To maintain the chip's passive operation, the photodetector circuitrymay use voltage supplied by the same RF signal that is used to writeother data to memory, so that no detection of photoemission will occurunless RF or other power is applied to provide bias and drain voltage.If an active device is used, the power supplied by the battery canprovide operational voltage to the photodetector circuitry, independentof any transmitted signal. The voltage supplied by the photodetector canbe used in a number of different ways. For example:

1) The threshold voltage for writing to memory will exceed the voltagesupplied by the RF signal, which will still contain the addressinformation. In order to write, additional voltage must be provided bythe photodetector so that the sum of the voltages exceeds the threshold.(V_(RF)<V_(T)<V_(RF)+V_(PD)). This permits the RF supplied voltage to goto the correct address, however, no writing will occur unless aphotoemission has been detected by the detector. Therefore, there willbe no record of exposure to a particular process step unless asufficient reaction has occurred to generate the required photoemission.Since the address signal can still get to the memory array without theextra voltage, reading of recorded data can be achieved without anyspecial circuitry. If the memory device is an active device, a similarmechanism can be used in which only the sum of the voltages issufficient to record an occurrence.

2) The threshold voltage for writing to memory will be provided by theRF signal alone, and the RF signal will include address information.(V_(T)<V_(RF)). Unless voltage from the photodetector is supplied to a“gating” transistor, however, access to the memory array is prevented sothat no writing occurs unless a photoemission is detected. (Thisembodiment is illustrated.) This will require a special provision foropening the gate during read operations to permit access to the memoryarray. Since the gating transistor will conduct a signal only in theevent of photoemission, this embodiment will work equally well withpassive and active memory devices.

3) The RF signal provides sufficient voltage to exceed the thresholdvoltage. (V_(T)<V_(RF)). Voltage from the photodetector is used tocreate a write potential difference at an additional address locationwhich is carried in the RF signal. For example, if the RF signal isaddressing column 3, row 3, column 32 could be connected only to thephotodetector circuit's output so that, when a photoemission occurs, thewrite signal will create antifuses [or in the case of EEPROM, standardfuses] at addresses 3,3 and 32,3. If no photoemission occurs, onlyaddress 3,3 will have an antifuse formed, providing a record of exposureof the matrix to a particular process step even without the occurrenceof a detectable reaction. Special provisions, such as software withinthe host computer in combination with mask-programmed interconnectionswithin the decode circuitry of the memory device, must be made to assurethat more than one column in a single row of the array is polled duringread operations so that both memory locations are read.

In addition to the above-described methods for recording the occurrenceof photo-emitting reactions, the photodetector, while still integratedon the same substrate with the basic memory matrix for recordingtransmitted signals, can be connected to its own independent memorymatrix. In this embodiment, the photodetector's memory matrix can beconnected to separate transceiver circuitry with an antenna tuned to adifferent frequency from that of the basic memory. During the readoperation, the memory device will be exposed to two different radiofrequency signals, one for the basic memory, the other for thephotodetection circuit memory. If only the photoemission information isrequired, only the corresponding frequency signal need be providedduring the read operation.

Depending on the type of energy release that occurs during a reaction,other types of sensors may be used in addition to photodetectors or inplace thereof. In addition changes in ion concentration may also bedetected. Many such sensors will be capable of generating an electricalsignal that can be used as described above for the photodetectors. Thesesensing devices may also be incorporated onto the substrate andelectrically connected to the memory device, providing data pointswithin the device's memory under the appropriate write conditions. Forexample, temperature sensing elements can be made from semiconductorliquid crystal and fluorescent crystals, and addition to conventionalthermocouples created by placing two different metals in contact at thedetection point. It is also possible to include radiation, pH and PCO₂sensors in a similar manner, using materials that respond to thedetected variables by generating a voltage potential that can beconducted to the memory device and recorded.

The reaction-detecting embodiment may be advantageously used in assays,such as the SPA, HTRF, FET, FRET and FP assays described below. In theseassays, reaction, such as receptor binding, produces a detectablesignal, such as light, in the matrix. If a matrix with memory with aphotodetection circuit is used, occurrence of the binding reaction willbe recorded in memory.

b. Devices for Drug Delivery and Sensors for Detecting Changes inInternal Conditions in the Body

Memories may also be combined with biocompatible supports and polymersthat are used internally in the bodies of animals, such as drug deliverydevices [see, e.g., U.S. Pat. Nos. 5,447,533, 5,443,953, 5,383,873,5,366,733, 5,324,324, 5,236,355, 5,114,719, 4,786,277, 4,779,806,4,705,503, 4,702,732, 4,657,543, 4,542,025, 4,530,840, 4,450,150 and4,351,337] or other biocompatible support [see, U.S. Pat. No. 5,217,743and U.S. Pat. No. 4,973,493, which provide methods for enhancing thebiocompatibility of matrix polymers). Such biocompatible polymersinclude matrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid [see,e.g., Sherwood et al. (1992) Bio/Technology 10:1446-1449].

The biocompatible drug delivery device in combination with the memory isintroduced into the body. The device, generally by virtue of combinationwith a biosensor or other sensor, also monitors pH, temperature,electrolyte concentrations and other such physiological parameters andin response to preprogrammed changes, directs the drug delivery deviceto release or not release drugs or can be queried, whereby the change isdetected and drug delivered or administered.

Alternatively, the device provided in combination with a biocompatiblesupport and biosensor, such that the information determined by thebiosensor can be stored in the device memory. The combination of deviceand biosensor is introduced into the body and is used to monitorinternal conditions, such as glucose level, which level is written tomemory. The internal condition, such as glucose level, electrolytes,particularly potassium, pH, hormone levels, and other such level, canthen be determined by querying the device.

In one embodiment, the device, such as one containing a memory that isread to and written using RF, linked to a biosensor [see, e.g., U.S.Pat. No. 5,384,028 which provides a biosensor with a data memory thatstores data] that can detect a change in an internal condition, such asglucose or electrolyte, and store or report that change via RF to thelinked matrix with memory, which records such change as a data point inthe memory, which can then be queried. The animal is then scanned withRF and the presence of the data point is indicative of a change. Thus,instead of sampling the body fluid, the memory with matrix with linkedbiosensor is introduced into a site in the body, and can be queriedexternally. For example, the sensor can be embedded under the skin andscanned periodically, or the scanner is worn on the body, such as on thewrist, and the matrix with memory either periodically, intermittently,or continuously sends signals; the scanner is linked to an infusiondevice and automatically, when triggered triggers infusion or altersinfusion rate.

A well-known problem that can rapidly render a sensing implantineffective is the body's natural response to “wall-off” encapsulate theimplant such that the concentration of any analyte in this poorlyvascularized tissue surrounding the implant does not properly reflectthe analyte concentration in tissue as a whole. A method for solvingthis problem is provided herein. Any implantable biosensor may bemodified by including an angiogenic material in the matrix thatsurrounds the implant. The angiogenic material will promote the growthof new vascularization into the tissue immediately surrounding theimplant and thus improve transport of analyte to the sensor implant.Examples of angiogenic factors, include, but are not limited to basicfibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF),epidermal growth factor (EGF), vascular endothelial growth factor(VEGF), platelet-derived growth factor (PDGF), heparin, prostaglandinE1, and interleukins, such as IL-1a and IL-8, among others.

Also, the implant may be coated with collagen and/or heparin to whichthe angiogenic factor, such as bFGF, is absorbed, thereby presenting thefactor in a configuration that mimics its in vivo presentation.

In addition, these factors may be provided in a time release format,such as by containing the angiogenic factor within a collagen gel inconjunction with, for example, an aluminum sucrose octasulfatesuspension.

4. Reading and Writing to Memory a. Embodiments Using a ProximateMemory, Such as a Non-volatile Memory Device

The operation of programming the memory to record the process steps towhich the linked or adjacent matrix particle or support and linked orproximate molecule or biological particle is exposed involves placingthe memory device reasonably close. A distance on the order of about 1inch (25.4 mm) to RF transmitter 50 is presently contemplated, butlonger distances should be possible and shorter distances are alsocontemplated. Suitable distances can be determined empirically. The RFtransmitter 50 emits a carrier wave modulated by a signal generated byhost computer 70 using conventional RF technology. The carrier waveitself can provide the power to the generate the programming voltage andthe operating voltage for the various devices via the rectifier, whilethe modulation signal provides the address instructions.

As stated previously, since the memory only has to be “tagged” to recordthe exposure of the proximate or linked molecule or biological particleto a given process, the address signal only has to carry information toturn on a single memory location, while the host computer 70 stores intomemory 68 the information linking the process information with thesingle memory location that was “tagged” to record exposure to theprocess step. Referring to FIG. 1, in which chemical building blocks A,C, and E are added to a molecule linked to a matrix with memory, and toFIG. 6, an illustrative example of how information is written onto aparticle is provided in Table 1.

TABLE 1 PROCESS STEP X-REGISTER ADDRESS Y-REGISTER ADDRESS A 1 8 C 2 4 E3 2

For the step in which A is added, the address signal would increment thex-register 124 one location and increment the y-register 126 eightlocations, and then apply the programming voltage. The activation ofthis switch is indicated by an “A” at the selected address, although theactual value stored will be a binary “1”, indicating ON. (As described,for example, in U.S. Pat. No. 4,424,579; the manner in which theprogramming voltage is applied depends on whether the decoders havedepletion or enhancement transistors.) The host computer 70 would writeinto its memory 148 that for process A, the x-,y- address is 1,8. Uponremoval of the RF signal after recording process A, the voltage isremoved and the registers would reset to 0. For the step in which C isadded, the address signal would increment the x-register 124 twolocations and the y-register 126 four locations, then apply theprogramming voltage, as indicated by the letter “C”. The host computer70 would similarly record in memory that an indication of exposure toprocess C would be found at x-,y- address 2,4. Again, upon removal ofthe RF signal, the registers reset to 0 so that when the matrixparticle's memory is again exposed to RF following addition of block E,the registers increment 3 and 2 locations, respectively, and theprogramming voltage is applied to turn on the switch, indicated by “E”.Desirably all processing steps are automated.

After processing is completed, to read the information that has beenrecorded in the memory of the data storage unit, the host computer 70will inquire into the identity of the particle by generating a commandsignal to the registers to select the appropriate address locations todetermine whether the switch is on or off. If the switch is on, i.e., avoltage drop occurs at that point, the computer will create a recordthat the particle received a particular process step. Alternatively, thehost computer can generate an inquiry signal to sequentially look at allmemory locations to determine which switches have been turned on,recording all locations at which voltage drops occurred. The computerwill then compare the “on” locations to the process steps stored in itsmemory to identify the steps through which the subject particle wasprocessed.

If desired, individual particles can be identified by reserving certainmemory locations for identification only, for example, the first tworows of the x-register. In this case, particles will be passedseparately through the RF signal while the x-register is incremented toturn on switches at address locations 0,0, 1,0, 2,0, etc. Withindividual identification, the host computer 70 can first generate asignal to query a matrix particle memory to determine its identity, thenwrite the information with regard to the process performed, saving theprocess and particle information in the host computer memory 68.

Ideally, the tagging of particles which are exposed to a particularprocess would be performed in the process vessel containing all of theparticles. The presence, however, of a large number of particles mayresult in interference or result in an inability to generate asufficiently high voltage for programming all of the particlessimultaneously. This might be remedied by providing an exposure ofprolonged duration, e.g., several minutes, while stirring the vesselcontents to provide the greatest opportunity for all particles toreceive exposure to the RF signal. On the other hand, since eachparticle will need to be read individually, a mechanism for separatingthe particles may be used in write and read operations. Also, ininstances in which each particle will have a different moleculeattached, each particle memory must be addressed separately.

b. Embodiments Using OMDs

When precoded OMDs are used, each OMD (or group thereof) has a uniqueidentifier is optically scanned and entered into a remote memory.Thereafter, after each synthesis, processing or assaying step,information regarding such for each device identified by its encodedsymbology is entered into a remote memory. Upon completion of thesynthesis, processing, assay or other protocol, each device can bescanned and identified. Reference to the information stored in theremote memory provides information regarding the linked molecules orbiological particles or the assay or other information. When read/writeOMDs are used, identifying symbology is encoded on the device and thedecrypting information is stored in a remote memory.

D. The combinations and preparation thereof

Combinations of a memory, such as an optical memory, a magnetic memoryor a miniature recording device, that contains or is a data storage unitlinked to or in proximity with matrices or supports used in chemical andbiotechnical applications, such as combinatorial chemistry, peptidesynthesis, nucleic acid synthesis, nucleic acid amplification methods,organic template chemistry, nucleic acid sequencing, screening fordrugs, particularly high throughput screening, phage display screening,cell sorting, drug delivery, tracking of biological particles and othersuch methods, are provided. These combinations of matrix material withdata storage unit [or recording device including the unit] are hereinreferred to as matrices with memories. These combinations have amultiplicity of applications, including combinatorial chemistry,isolation and purification of target macromolecules, capture anddetection of macromolecules for analytical purposes, high throughputscreening protocols, selective removal of contaminants, enzymaticcatalysis, drug delivery, chemical modification, scintillation proximityassays, FET, FRET and HTRF assays, immunoassays, receptor bindingassays, drug screening assays, information collection and management andother uses. These combinations are particularly advantageous for use inmultianalyte analyses. These combinations may also be advantageouslyused in assays in which a electromagnetic signal is generated by thereactants or products in the assay. These combinations may be used inconjunction with or may include a sensor element, such as an elementthat measures a solution parameter, such as pH. Change in suchparameter, which is recorded in the memory will indicate a reactionevent of interest, such as induction of activity of a receptor or ionchannel, has occurred. The combination of matrix with memory is alsoadvantageously used in multiplex protocols, such as those in which amolecule is synthesized on the matrix, its identity recorded in thematrix, the resulting combination is used in an assay or in ahybridization reaction. Occurrence of the reaction can be detectedexternally, such as in a scintillation counter, or can be detected by asensor that writes to the memory in the matrix. Thus, combinations ofmatrix materials, memories, and linked or proximate molecules andbiological materials and assays using such combinations are provided.

The combinations contain (i) a miniature recording device that containsone or more programmable data storage devices [memories] that can beremotely read and in preferred embodiments also remotely programmed; and(ii) a matrix as described above, such as a particulate support used inchemical syntheses. The remote programming and reading is preferablyeffected using electromagnetic radiation, particularly radio frequencyor radar, microwave, X-rays. Depending upon the application thecombinations will include additional elements, such as scintillants,photodetectors, pH sensors and/or other sensors, and other suchelements.

1. Preparation of Matrix-memory Combinations

In preferred embodiments, the recording device is cast in a selectedmatrix material during manufacture. Alternatively, the devices can bephysically inserted into the matrix material, the deformable gel-likematerials, or can be placed on the matrix material and attached by aconnector, such as a plastic or wax or other such material.Alternatively, the device or device(s) may be included in an inertcontainer in proximity to or in contact with matrix material.

2. Non-linked matrix-memory Combinations

The recording device with memory can be placed onto the inner or outersurface of a vessel, such as a microtiter plate or vial or tube in whichthe reaction steps are conducted, fractions collected or samples stored.Alternatively, the device can be incorporated into the vessel material,such into the a wall of each microtiter well or vial or tube in whichthe reaction is conducted. As long as the molecules or biologicalparticles remain associated with the well, tube or vial, such as a vialused to collect HPLC fractions, or a vial containing a patient sample,their identity can be tracked. The memory will be a programmableelectronic memory or a bar code. These memories can also be associatedwith reagent containers.

A bar code reader, transponder reader or other such device can be usedto enter the desired information building block name by reading thereagent container. Such information will be entered in to matrix memoryor remote computer memory. Software for doing so can be integrated intothe systems used, such as the bar code reader described herein or thetransponders and encoders described herein.

Also of interest herein are the multiwell “chips” [such as thoseavailable from Orchid Biocomputer, Inc. Princeton, N.J., see, e.g., U.S.Pat. Nos. 5,047,371, 4,952,531, 5,043,222, 5,277,724, 5,256,469 andPrabhu et al. (1992) Proc. SPIE-Int. Soc. Opt. Eng. 1847 NUMBER:Proceedings of the 1992 International Symposium on Microelectronics,pp.601-6, that are silicone based chips that contain 10,000 microscopicwells connected by hair-thin glass tubes to tiny reservoirs containingreagents for synthesis of compounds in each well. Each well can bemarked with a code and the code associated with the identity of thesynthesized compound in each well. Ultimately, a readable or read/writememory may be incorporated into each well, thus permitting rapid andready identification of the contents of each well.

In a particularly preferred embodiment, one or more recording deviceswith memory and matrix particles are sealed in a porous non-reactivematerial, such as polypropylene or TEFLON net, with a pore size smallerthan the particle size of the matrix and the device. Typically onedevice per about 1 to 50 mg, preferably 5 to 30, more preferably 5 to 20mg of matrix material, or in some embodiments up to gram, generally 50to 250 mg, preferably 150 mg to about 200 mg, and one device is sealedin a porous vessel a microvessel [MICROKAN™]. The amount of matrixmaterial is a function of the size of the device and the application inwhich the resulting matrix with memory is used, and, if necessary can beempirically determined. Generally, smaller sizes are desired, and theamount of material will depend upon the size of the selected recordingdevice.

The resulting microvessels are then encoded, reactions, such assynthetic reactions, performed, and read, and if desired used in desiredassays or other methods.

3. Combinations of Memories With Sensors and Matrices With Memories andSensors

As discussed above, below, and exemplified below, combinations ofsensors with memories and sensors with matrices with memories areprovided. Also provided are sol-gels with memories, coated sensors andother embodiments.

4. Preparation of Matrix-memory-molecule or Biological ParticleCombinations

In certain embodiments, combinations of matrices with memories andbiological particle combinations are prepared. For example, libraries[e.g., bacteria or bacteriophage, or other virus particles or otherparticles that contain genetic coding information or other information]can be prepared on the matrices with memories, and stored as such forfuture use or antibodies can be linked to the matrices with memories andstored for future use.

5. Combinations For Use in Proximity Assays

In other embodiments the memory or recording device is coated orencapsulated in a medium, such as a gel, that contains one or morefluophors or one or more scintillants, such as 2,5-diphenyloxazole [PPO]and/or 1,4-bis-[5-phenyl-(oxazolyl)]benzene [POPOP] or FlexiScint [a gelwith scintillant available from Packard, Meriden, Conn.] or yttriumsilicates. Any fluophore or scintillant or scintillation cocktail knownto those of skill in the art may be used. The gel coated or encaseddevice is then coated with a matrix suitable, such as glass orpolystyrene, for the intended application or applications. The resultingdevice is particularly suitable for use as a matrix for synthesis oflibraries and subsequent use thereof in scintillation proximity assays.

Similar combinations in non-radioactive energy transfer proximityassays, such as HTRF, FP, FET and FRET assays, which are describedbelow. These luminescence assays are based on energy transfer between adonor luminescent abel, such as a rare earth metal cryptate [e.g., Eutrisbipyridine diamine (EuTBP) or Tb tribipyridine diamine (TbTBP)] andan acceptor luminescent label, such as, when the donor is EuTBP,allopycocyanin (APC), allophycocyanin B, phycocyanin C or phycocyanin R,and when the donor is TbTBP, a rhodamine, thiomine, phycocyanin R,phycoerythrocyanin, phycoerythrin C, phycoerythrin B or phycoerythrin R.Instead of including a scintillant in the combination, a suitablefluorescent material, such as allopycocyanin (APC), allophycocyanin B,phycocyanin C, phycocyanin R; rhodamine, thiomine, phycocyanin R,phycoerythrocyanin, phycoerythrin C, phycoerythrin B or phycoerythrin Ris included. Alternatively, a fluorescent material, such a europiumcryptate is incorporated in the combination.

6. 2-D Bar Codes, Other Symbologies and Application Thereof

Any application and combination described herein in which a recordingdevice in proximity with a matrix may include a code or symbology inplace of or in addition to the recording device. The informationassociated with the code is stored in a remote recording device, such asa computer. Thus, by electro-optically scanning the symbol on thecombination and generating a corresponding signal, it is possible in anassociated computer whose memory has digitally stored therein the fullrange of codes, to compare the signal derived from the scanned symbolwith the stored information. When a match is found, the identity of theitem and associated information, such as the identity of the linkedmolecule or biological particle or the synthetic steps or assayprotocol, can be retrieved.

The symbology can be engraved on any matrix used as a solid support forchemical syntheses, reactions, assays and other uses set forth herein,for identification and tracking of the linked or proximate biologicalparticles and molecules. Particularly preferred is the two-dimensionalbar code and system used therewith for reading and writing the codes onmatrix materials.

7. Other Variations and Embodiments

The combination of matrix particle with memory may be further linked,such as by welding using a laser or heat, to an inert carrier or othersupport, such as a TEFLON strip. This strip, which can be of anyconvenient size, such as 1 to 10 mm by about 10 to 100 μM will renderthe combination easy to use and manipulate. For example, these memorieswith strips can be introduced into 10 cm culture dishes and used inassays, such as immunoassays, or they can be used to introduce bacteriaor phage into cultures and used in selection assays. The strip may beencoded or impregnated with a bar code to further provide identifyinginformation.

Microplates containing a recording device in one or a plurality of wellsare provided. The plates may further contain embedded scintillant or acoating of scintillant [such as FlashPlate™, available from DuPont NEN™,Cytostar-T plates from Amersham International plc, U.K., and platesavailable from Packard, Meriden, CT] FLASHPLATE™ is a 96 well microplatethat is precoated with plastic scintillant for detection of β-emittingisotopes, such as ¹²⁵I, ³H, ³⁵S, ¹⁴C and ³³P. A molecule is immobilizedor synthesized in each well of the plate, each memory is programmed withthe identify of each molecule in each well. The immobilized molecule onthe surface of the well captures a radiolabeled ligand in solutionresults in detection of the bound radioactivity. These plates can beused for a variety of radioimmmunoassays [RIAs], radioreceptor assays[RRAs], nucleic acid/protein binding assays, enzymatic assays andcell-based assays, in which cells are grown on the plates.

Another embodiment is depicted in FIG. 19. The reactive sites, such asamines, on a support matrix [1 in the FIGURE] in combination with amemory [a MICROKAN™, a MICROTUBE™, a MACROBEAD™, a MICROCUBE™ or othermatrix with memory combination] are differentiated by reacting them witha selected reaction of Fmoc-glycine and Boc-glycine, thereby producing adifferentiated support [2]. The Boc groups on 2 are then deprotectedwith a suitable agent such as TFA, to produce 3. The resulting fee aminegroups are coupled with a fluophore [or mixture A and B], to produce afluorescent support 4, which can be used in subsequent syntheses or forlinkage of desired molecules or biological particles, and then used influorescence assays and SPAs.

E. The recording and reading and systems

Systems for recording and reading information are provided. The systemsinclude a host computer or decoder/encoder instrument, a transmitter, areceiver and the data storage device. The systems also can include afunnel-like device and other sorting devices for use in separatingand/or tagging single memory devices. In practice, an EM signal or othersignal is transmitted to the data storage device. The antenna or otherreceiver means in the device detects the signal and transmits it to thememory, whereby the data are written to the memory and stored in amemory location. As provided herein, methods for addressing individualmemories in a batch are provided, thereby eliminating the need forsorting.

Mixtures of the matrix with memory-linked molecules or biologicalparticles may be exposed to the EM signal, or each matrix with memory[either before, after or during linkage of the biological particles ormolecules] may be individually exposed, using a device, such as thatdepicted herein, to the EM signal. Each matrix with memory, as discussedbelow, will be linked to a plurality of molecules or biologicalparticles, which may be identical or substantially identical or amixture of molecules or biological particles depending, upon theapplication and protocol in which the matrix with memory and linked [orproximate] molecules or biological particles is used. The memory can beprogrammed with data regarding such parameters.

The location of the data, which when read and transmitted to the hostcomputer or decoder/encoder instrument, corresponds to identifyinginformation about linked or proximate molecules or biological particles.The host computer or decoder/encoder instrument can either identify thelocation of the data for interpretation by a human or another computeror the host computer or the decoder/encoder can be programmed with a keyto interpret or decode the data and thereby identify the linked moleculeor biological particle.

As discussed above, the presently preferred system for use is theIPTT-100 transponder and DAS-5001 CONSOLE™ [Bio Medic Data Systems,Inc., Maywood, N.J.; see, e.g., U.S. Pat. Nos. 5,422,636, 5,420,579,5,262,772, 5,252,962 and 5,250,962, 5,252,962 and 5,262,772].

These systems may be automated or may be manual.

F. Manual system

The manual system includes a memories with matrices, a device forreading and writing thereto and software therefor. The presentlypreferred manual system in which the matrices with memories haveelectromagnetically programmable memories, includes a transponder,particularly the BMDS transponder described below or an IDTAGtransponder or the monolithic chip provided herere, or any suitable reador read/write memory device and uses the corresponding reading andwriting device, which has been reconfigured and repackaged, such as inFIG. 17, described in the EXAMPLES. An example of the operation of thesystem of FIG. 17 is illustrated in FIG. 18 and described in EXAMPLE 4.Briefly, the user manually places a microvessel 180 with memory withinthe recessed area 176 so that the interrogation signal 185 provides aresponse to the controllers indicating the presence on the microvessel,and information is read from or written to the transponder.,

This will include microvessels, such as MICROKANS™ or MICROTUBES™,MICROBEADS™, MICROBALS™ read/writer hardware [such as that availablefrom BMDS or IDTAG™] connected to a PC and software running on the PCthat performs a user interface and system control function. The softwareis designed to facilitate the a number of aspects of syntheticcombinatorial chemistry libraries, including: organization, planning anddesign, synthesis compound formula determination, molecular weightcomputation, reporting of plans, status and results.

In particular, for each chemical library, the software creates a database file. This file contains all of the information pertinent to thelibrary, including chemical building blocks to be used, the design ofthe library in terms of steps and splits, and what synthesis has beenperformed. This file oriented approach allows many different chemicallibrary projects to be conducted simultaneously. The software allows theuser to specify what chemical building blocks are to be used and theirmolecular weights. The user specifies the number of steps, the number of“splits” at each step, and what chemical building blocks are to be usedat each split. The user may also enter the name of the pharmacophore andits molecular weight. Additionally, the user may specify graphicalchemical diagrams for the building blocks and the pharmacophore. Thisinformation is useful in displaying resulting compounds. The softwarerecords all of the above “design” information. It computes and displaysthe size of the library. It may also predict the range of molecularweights of the resulting compounds.

For example, the user specifies that there will be eight chemicalbuilding blocks. Their names are entered, and the user enters a uniqueletter codes for each: A, B, C, D, E, F, G and H. The user specifiesthat there will be three steps. Step one will have four splits,appending the A, B, C and D building blocks. Step two will also havefour splits, adding the B, D, E and H building blocks. Step three willhave six splits, adding the B, C, D, E, F and G building blocks. Thesoftware computes that the library will contain 96 (4×6×5=96) uniquecompounds. With the planning and design completed, the software helpsthe user perform the synthesis steps. This is done in concert with thereader/writer hardware [transceiver or a scanner, such as the BMDS—DAS5003] or a similar device available form IDTAG Ltd [Bracknell, BerksRG12 3XQ, UK] and devices, such as the MICROKAN™ or MICROTUBE™microvessel with memory devices. Before the synthesis begins, themicrovessels are filled with polymer resin. The microvessel devices are,one at a time placed upon the scanner. The device and software reads thecontents of the data encoded in the recording device, transponder, suchas the BMDS tag or the IDTAG™ tag, contained in each microvessel. Thesoftware, chooses which building block shall be added to the compoundcontained in each microvessel. It directs the transceiver to writeencoded data to the transponder, indicating which building block thisis. The software displays a message which directs the user to place themicrovessel in the appropriate reaction vessel so that the chosenbuilding block will be added. This process is repeated a plurality oftimes with each microvessel and for each synthetic step the plannedsteps of the library.

The software then uses the scanner to read a tag and receive its encodedinformation. Using the user-entered compound names stored in thelibrary's data base, the software translates the encoded informationinto the names of the chemical building blocks. The software can alsodisplay compounds graphically, using the graphical information specifiedby the user. The software calculates the molecular weight of compoundsfrom the data provided for the pharmacophore and building blocks.

The software facilitates the recording of progress through the aboveprocess. The software generates displays and reports which illustratethis and all of the above planning, design, compound data, and graphicalrepresentations of compounds. An example of the software [to becommercialized under the name Synthesis Manager] and use thereof isdescribed in the Examples, below. Once armed with the instantdisclosure, other such software can be developed by one skilled in thatart.

Briefly, in the first step in building a library, the individualbuilding blocks [i.e., the monomers, nucleotides or amino acids or othersmall molecules] and the steps in which they will be used are defined.The software then automatically creates a data base record for eachcompound to be synthesized. Pre-reaction procedures, reactionconditions, and work up procedures are stored for each step.

When the synthesis begins, the step “Perform Synthesis” is selected. Thesoftware plays back to the user the procedure, and then reads each ofthe memories in each microreactor and sorts them for the next reactionstep. When the sorting is complete, the reaction condition informationand work-up procedure are also displayed to the user.

When the chemical synthesis is complete, compounds are cleaved from themicroreactors and archived. The software provides archival capabilityfor either individual vials or a 96-well format or will be adapted forother formats. Specific columns, rows, or individual well scan beprotected to accommodate the need for standards and controls invirtually any screening format.

The software provides several utilities that permit one tag to be readat any time, display the corresponding building block names andstructures, and the current synthesis status of that compound. It isalso possible to search for a specific compound or compounds thatcontain certain building blocks. For compounds that have already beenarchived, the archive location [i.e., microplate group name, number, andwell] will be displayed.

G. Tools and Applications using matrices with memories

1. Tools

The matrix with memory and associated system as described herein is thebasic tool that can be used in a multitude of applications, includingany reaction that incorporates a functionally specific (i.e. in thereaction) interaction, such as receptor binding. This tool is thencombined with existing technologies or can be modified to produceadditional tools.

For example, the matrix with memory combination, can be designed as asingle analyte test or as a multianalyte test and also as a multiplexedassay that is readily automated. The ability to add one or a mixture ofmatrices with memories, each with linked or proximate molecule orbiological particle to a sample, provides that ability to simultaneouslydetermine multiple analytes and to also avoid multiple pipetting steps.The ability to add a matrix with memory and linked molecules orparticles with additional reagents, such as scintillants, provides theability to multiplex assays.

As discussed herein, in one preferred embodiment the matrices areparticulate and include adsorbed, absorbed, or otherwise linked orproximate, molecules, such as peptides or oligonucleotides, orbiological particles, such as cells. Assays using such particulatememories with matrices may be conduced “on bead” or “off bead”. On beadassays are suitable for multianalyte assays in which mixtures ofmatrices with linked molecules are used and screened against a labeledknown. Off bead assays may also be performed; in these instances theidentity of the linked molecule or biological particle must be knownprior to cleavage or the molecule or biological particle must be in somemanner associated with the memory.

In other embodiments the matrices with memories use matrices that arecontinuous, such as microplates, and include a plurality of memories,preferably one memory/well. Of particular interest herein are matrices,such as Flash Plates™ [NEN, Dupont], that are coated or impregnated withscintillant or fluophore or other luminescent moiety or combinationthereof, modified by including a memory in each well. The resultingmatrix with memory is herein referred to as a luminescing matrix withmemory. Other formats of interest that can be modified by including amemory in a matrix include the Multiscreen Assay System [Millipore] andgel permeation technology. Again it is noted that the memories may bereplaced with or supplemented with engraved code, preferably at the baseof each well [outer surface preferred] that is either precoded or addedprior to or during use. The memory, in these instances, is then remotefrom the matrix.

Preferred plates are those that contain a microplate type frame andremovable wells or strips. Each well or strip can contain a memoryand/or can be engraved with a code.

2. Scintillation Proximity Assays (SPAs) and Scintillant-containingMatrices with Memories

Scintillation proximity assays are well known in the art [see, e.g.,U.S. Pat. No. 4,271,139; U.S. Pat. No. 4,382,074; U.S. Pat. No.4,687,636; U.S. Pat. No. 4,568,649; U.S. Pat. No. 4,388,296; U.S. Pat.No. 5,246,869; International PCT Application No. WO 94/26413;International PCT Application No. WO 90/03844; European PatentApplication No. 0 556 005 A1; European Patent Application No. 0 301 769A1; Hart et al. (1979) Molec. Immunol. 16:265-267; Udenfriend et al.(1985) Proc. Natl. Acad. Sci. U.S.A. 82:8672-8676; Nelson et al. (1987)Analyt. Biochem 165:287-293; Heath, et al. (1991) Methodol. Surv.Biochem. Anal. 21:193-194; Mattingly et al. (1995) J. Memb. Sci.98:275-280; Pernelle (1993) Biochemistry 32:11682-116878; Bosworth etal. (1989) Nature 341:167-168; and Hart et al. (1989) Nature 341:265].Beads [particles] and other formats, such as plates and membranes havebeen developed.

SPA assays refer to homogeneous assays in which quantifiable lightenergy produced and is related to the amount of radioactively labelledproducts in the medium. The light is produced by a scintillant that isincorporated or impregnated or otherwise a part of a support matrix. Thesupport matrix is coated with a receptor, ligand or other capturemolecule that can specifically bind to a radiolabeled analyte, such as aligand.

a. Matrices

Typically, SPA uses fluomicrospheres, such as diphenyloxazole-latex,polyacrylamide-containing a fluophore, and polyvinyltoluene [PVT]plastic scintillator beads, and they are prepared for use by adsorbingcompounds into the matrix. Also fluomicrospheres based on organicphosphors have been developed. Microplates made from scintillationplastic, such as PVT, have also been used [see, e.g., International PCTApplication No. WO 90/03844]. Numerous other formats are presentlyavailable, and any format may be modified for use herein by includingone or more recording devices.

Typically the fluomicrospheres or plates are coated with acceptormolecules, such as receptors or antibodies to which ligand bindsselectively and reversibly. Initially these assays were performed usingglass beads containing fluors and functionalized with recognition groupsfor binding specific ligands [or receptors], such as organic molecules,proteins, antibodies, and other such molecules. Generally the supportbodies used in these assays are prepared by forming a porous amorphousmicroscopic particle, referred to as a bead [see, e.g., European PatentApplication No.0 154,734 and International PCT Application No. WO91/08489]. The bead is formed from a matrix material such as acrylamide,acrylic acid, polymers of styrene, agar, agarose, polystyrene, and othersuch materials, such as those set forth above. Cyanogen bromide has beenincorporated into the bead into to provide moieties for linkage ofcapture molecules or biological particles to the surface. Scintillantmaterial is impregnated or incorporated into the bead by precipitationor other suitable method. Alternatively, the matrices are formed fromscintillating material [see, e.g., International PCT Application No. WO91/08489, which is based on U.S. application Ser. No. 07/444,297; see,also U.S. Pat. No. 5,198,670], such as yttrium silicates and otherglasses, which when activated or doped respond as scintillators. Dopantsinclude Mn, Cu, Pb, Sn, Au, Ag, Sm, and Ce. These materials can beformed into particles or into continuous matrices. For purposes herein,the are used to coat, encase or otherwise be in contact with one or aplurality of recording devices.

Assays are conducted in normal assay buffers and requires the use of aligand labelled with an isotope, such as ³H and ¹²⁵I, that emitslow-energy radiation that is readily dissipated easily an aqueousmedium. Because ³H β particles and ¹²⁵I Auger electrons have averageenergies of 6 and 35 keV, respectively, their energies are absorbed bythe aqueous solutions within very small distances (˜4 μm for ³H βparticles and 35 Arm for ¹²⁵I Auger electrons). Thus, in a typicalreaction of 0.1 ml to 0.4 ml the majority of unbound labelled ligandswill be too far from the fluoromicrosphere to activate the fluor. Boundligands, however, will be in sufficiently close proximity to thefluomicrospheres to allow the emitted energy to activate the fluor andproduce light. As a result bound ligands produce light, but free ligandsdo not. Thus, assay beads emit light when they are exposed to theradioactive energy from the label bound to the beads through theantigen-antibody linkage, but the unreacted radioactive species insolution is too far from the bead to elicit light. The light from thebeads will be measured in a liquid scintillation counter and will be ameasure of the bound label.

Matrices with memories for use in scintillation proximity assays [SPA]are prepared by associating a memory [or engraved or imprinted code orsymbology) with a matrix that includes a scintillant. In the most simpleembodiment, matrix particles with scintillant [fluomicrospheres] arepurchased from Amersham, Packard, NE Technologies [(formerly NuclearEnterprises, Inc.) San Carlos, Calif.] or other such source and areassociated with a memory, such as by including one or more of such beadsin a MICROKAN™ microvessel with a recording device. Typically, suchbeads as purchased are derivatized and coated with selected moieties,such as streptavidin, protein A, biotin, wheat germ agglutinin [WGA],and polylysine. Also available are inorganic fluomicrospheres based oncerium-doped yttrium silicate or polyvinyltoluene (PVT). These containscintillant and may be coated and derivatized.

Alternatively, small particles of PVT impregnated with scintillant areused to coat recording devices, such as the IPTT-100 devices [Bio MedicData Systems, Inc., Maywood, N.J.; see, also U.S. Pat. Nos. 5,422,636,5,420,579, 5,262,772, 5,252,962, 5,250,962, 5,074,318, and RE 34,936]that have been coated with a protective material, such as polystyrene,TEFLON, a ceramic or anything that does not interfere with the readingand writing EM frequency(ies). Such PVT particles may be manufactured orpurchased from commercial sources such as NE TECHNOLOGY, INC. [e.g.,catalog #191A, 1-10 μm particles]. These particles are mixed withagarose or acrylamide, styrene, vinyl or other suitable monomer thatwill polymerize or gel to form a layer of this material, which is coatedon polystyrene or other protective layer on the recording device. Thethickness of the layers may be empirically determined, but they must besufficiently thin for the scintillant to detect proximate radiolabels.To make the resulting particles resistant to chemical reaction they maybe coated with polymers such as polyvinyltoluene or polystyrene, whichcan then be further derivatized for linkage and/or synthesis ofmolecules and biological particles. The resulting beads are hereincalled luminescing matrices with memories, and when used in SPA formatsare herein referred to as scintillating matrices with memories.

The scintillating matrices with memories beads can be formed bymanufacturing a bead containing a recording device and includingscintillant, such as 2,5-diphenyloxazole [PPO] and/or1,4-bis-[5-phenyl-(oxazolyl)]benzene [POPOP] as a coating. Theseparticles or beads are then coated with derivatized polyvinyl benzene orother suitable matrix on which organic synthesis, protein synthesis orother synthesis can be performed or to which organic molecules,proteins, nucleic acids, biological particles or other such materialscan be attached. Attachment may be effected using any of the methodsknown to those of skill in the art, including methods described herein,and include covalent, non-covalent, direct and indirect linkages.

Alternatively or additionally, each bead may be engraved with a code.Preferably the beads are of such geometry that they can be readilyoriented for reading.

Molecules, such as ligands or receptors or biological particles arecovalently coupled thereto, and their identity is recorded in thememory. Alternatively, molecules, such as small organics, peptides andoligonucleoties, are synthesized on the beads as described herein sothat history of synthesis and/or identity of the linked molecule isrecorded in the memory. The resulting matrices with memory particleswith linked molecules or biological particles may be used in anyapplication in which SPA is appropriate. Such applications, include, butare not limited to: radioimmunoassays, receptor binding assays, enzymeassays and cell biochemistry assays.

For use herein, the beads, plates and membranes are either combined witha recording device or a plurality of devices, or the materials used inpreparing the beads, plates or membranes is used to coat, encase orcontact a recording device and/or engraved with a code. Thus,microvessels [MICROKANS™] containing SPA beads coated with a molecule orbiological particle of interest; microplates impregnated with or coatedwith scintillant, and recording devices otherwise coated with,impregnated with or contacted with scintillant are provided.

To increase photon yield and remove the possibility of loss of fluor,derivatized fluomicrospheres based on yttrium silicate, that is dopedselectively with rare earth elements to facilitate production of lightwith optimum emission characteristics for photomultipliers andelectronic circuitry have been developed [see, e.g., European PatentApplication No. 0 378 059 B1; U.S. Pat. No. 5,246,869]. In practice,solid scintillant fibers, such as cerium-loaded glass or based on rareearths, such as yttrium silicate, are formed into a matrix. The glassesmay also include activators, such as terbium, europium or lithium.Alternatively, the fiber matrix may be made from a scintillant loadedpolymer, such as polyvinyltoluene. Molecules and biological particlescan be adsorbed to the resulting matrix.

For use herein, these fibers may be combined in a microvessel with arecording device [i.e., to form a MICROKAN™]. Alternatively, the fibersare used to coat a recording device or to coat or form a microplatecontaining recording devices in each well. The resulting combinationsare used as supports for synthesis of molecules or for linkingbiological particles or molecules. The identity and/or location and/orother information about the particles is encoded in the memory and theresulting combinations are used in scintillation proximity assays.

Scintillation plates [e.g., FlashPlates™, NEN Dupont, and other suchplates] and membranes have also been developed [see, Mattingly et al.(1995) J. Memb. Sci. 98:275-280] that may be modified by including amemory for use as described herein. The membranes, which can containpolysulfone resin M.W. 752 kD, polyvinylpyrrolidone MW 40 kDA,sulfonated polysulfone, fluor, such as p-bis-o-methylstyrylbenzene, POPand POPOP, may be prepared as described by Mattingly, but used to coat,encase or contact a recording device. Thus, instead of applying thepolymer solution to a glass plate the polymer solution is applied to therecording device, which, if need is pre-coated with a protectivecoating, such as a glass, TEFLON or other such coating.

Further, as shown in the Examples, the recording device may be coatedwith glass, etched and the coated with a layer of scintillant. Thescintillant may be formed from a polymer, such as polyacrylamide,gelatin, agarose or other suitable material, containing fluophors, ascintillation cocktail, FlexiScint [Packard Instrument Co., Inc.,Downers Grove, Ill.] NE Technology beads [see, e.g., U.S. Pat. No.4,588,698 for a description of the preparation of such mixtures].Alternatively, microplates that contain recording devices in one or morewells may be coated with or impregnated with a scintillant ormicroplates containing scintillant plastic may be manufactured withrecording devices in each well. If necessary, the resulting bead,particle or continuous matrix, such as a microplate, may be coated witha thin layer polystyrene, TEFLON or other suitable material. In allembodiments it is critical that the scintillant be in sufficientproximity to the linked molecule or biological particle to detectproximate radioactivity upon interaction of labeled molecules or labeledparticles with the linked molecule or biological particle.

The resulting scintillating matrices may be used in any application forwhich scintillation proximity assays are used. These include, ligandidentification, single assays, multianalyte assays, includingmulti-ligand and multi-receptor assays, radioimmunoassays [RIAs], enzymeassays, and cell bio-chemistry assays [see, e.g., International PCTApplication No. WO 93/19175, U.S. Pat. No. 5,430,150, Whitford et al.(1991) Phytochemical Analysis 2:134-136; Fenwick et al. (1994) Anal.Proc. Including Anal. Commun. 31:103-106; Skinner et al. (1994) Anal.Biochem. 223:259-265; Matsumura et al. (1992) Life Sciences51:1603-1611; Cook et al. (1991) Structure and Function of the AsparticProteinases, Dunn, ed., Penum Press, NY, pp. 525-528; Bazendale et al.in (1990) Advances in Prostaglandin, Thromboxane and LeukotrieneResearch, Vol. 21, Samuelsson et al., eds., Raven Press, NY, pp302-306].

b. Assays (1) Receptor Binding Assays

Scintillating matrices with memories beads can be used, for example, inassays screening test compounds as agonists or antagonists of receptorsor ion channels or other such cell surface protein. Test compounds ofinterest are synthesized on the beads or linked thereto, the identity ofthe linked compounds is encoded in the memory either during or followingsynthesis, linkage or coating. The scintillating matrices with memoriesare then incubated with radiolabeled [¹²⁵I, ³H, or other suitableradiolabel] receptor of interest and counted in a liquid scintillationcounter. When radiolabeled receptor binds to any of the structure(s)synthesized or linked to the bead, the radioisotope is in sufficientproximity to the bead to stimulate the scintillant to emit light. Incontrast, if a receptor does not bind, less or no radioactivity isassociated with the bead, and consequently less light is emitted. Thus,at equilibrium, the presence of molecules that are able to bind thereceptor may be detected. When the reading is completed, the memory ineach bead that emits light [or more light than a control] queried andthe host computer, decoder/encoder, or scanner can interpret the memoryin the bead and identify the active ligand.

(a) Multi-ligand Assay

Mixtures of scintillating matrices with memories with a variety oflinked ligands, which were synthesized on the matrices or linked theretoand their identities encoded in each memory, are incubated with a singlereceptor. The memory in each light-emitting scintillating matrix withmemory is queried and the identity of the binding ligand is determined.

(b) Multi-receptor Assays

Similar to conventional indirect or competitive receptor binding assaysthat are based on the competition between unlabelled ligand and a fixedquantity of radiolabeled ligand for a limited number of binding sites,the scintillating matrices with memories permit the simultaneousscreening of a number of ligands for a number of receptor subtypes.

Mixtures of receptor coated beads [one receptor type/per bead; eachmemory encoded with the identity of the linked receptor] are reactedwith labeled ligands specific for each receptor. After the reaction hasreached equilibrium, all beads that emit light are reacted with a testcompound. Beads that no longer emit light are read.

For example receptor isoforms, such as retinoic acid receptor isoforms,are each linked to a different batch of scintillating matrix with memorybeads, and the identity of each isoform is encoded in the memories oflinked matrices. After addition of the radiolabeled ligand(s), such as³H-retinoic acid, a sample of test compounds [natural, synthetic,combinatorial, etc.] is added to the reaction mixture, mixed andincubated for sufficient time to allow the reaction to reachequilibrium. The radiolabeled ligand binds to its receptor, which hasbeen covalently linked to the bead and which the emitted short rangeelectrons will excite the fluophor or scintillant in the beads,producing light. When unlabelled ligand from test mixture is added, ifit displaces the labeled ligand it will diminish or stop the fluorescentlight signal. At the end of incubation period, the tube can be measuredin a liquid scintillation counter to demonstrate if any of the testmaterial reacted with receptor family. Positive samples [reduced or nofluorescence] will be further analyzed for receptor subtyping byquerying their memories with the RF detector. In preferred embodiments,each bead will be read and with a fluorescence detector and RF scanner.Those that have a reduced fluorescent signal will be identified and thelinked receptor determined by the results from querying the memory.

The same concept can be used to screen for ligands for a number ofreceptors. In one example, FGF receptor, EGF receptor, and PDGF receptorare each covalently linked to a different batch of scintillating matrixwith memory beads. The identity of each receptor is encoded in eachmemory. After addition of the ¹²⁵I-ligands [¹²⁵I-FGF, ¹²⁵I-EGF, and¹²⁵I-PDGF] a sample of test compounds [natural, synthetic,combinatorial, etc.) is added to the tube containing¹²⁵I-ligand-receptor-beads, m mixed and incubated for sufficient time toallow the reaction to reach equilibrium. The radiolabeled ligands bindto their respective receptors receptor that been covalently linked tothe bead. By virtue of proximity of the label to the bead, the emittedshort range electrons will excite the fluophor in the beads. Whenunlabelled ligand from test mixture is added, if it displaces the any ofthe labeled ligand it will diminish or stop the fluorescent signal. Atthe end of incubation period, the tube can be measured in a liquidscintillation counter to demonstrate if any of the test material reactedwith the selected receptor family. Positive samples will be furtheranalyzed for receptor type by passing the resulting complexes measuringthe fluorescence of each bead and querying the memories by exposing themto RF or the selected EM radiation. The specificity of test ligand isdetermined by identifying beads with reduced fluorescence that anddetermining the identity of the linked receptor by querying the memory.

(c) Other Formats

Microspheres, generally polystyrene typically about 0.3 μm-3.9 μm, aresynthesized with scintillant inside can either be purchased or preparedby covalently linking scintillant to the monomer prior to polymerizationof the polystyrene or other material. They can then be derivatized [orpurchased with chemical functional groups], such as —COOH, and —CH₂OH.Selected compounds or libraries are synthesized on the resultingmicrospheres linked via the functional groups, as described herein, orreceptor, such as radiolabeled receptor, can be coated on themicrosphere. The resulting “bead” with linked compounds, can used in avariety of SPA and related assays, including immunoassays, receptorbinding assays, protein:protein interaction assays, and other suchassays in which the ligands linked to the scintillant-containingmicrospheres are reacted with memories with matrices that are coatedwith a selected receptor.

For example, ¹²⁵I-labeled receptor is passively coated on the memorywith matrix and then mixed with ligand that is linked to a thescintillant-containing microspheres. Upon binding the radioisotope intois brought into close proximity to the scintillant in which effectiveenergy transfer from the β particle will occur, resulting in emission oflight.

Alternatively, the memory with matrix [containing scintillant] can alsobe coated with ³H-containing polymer on which the biological target[i.e., receptor, protein, antibody, antigen] can be linked [viaadsorption or via a functional group]. Binding of the ligand brings thescintillant into close proximity to the label, resulting in lightemission.

(2) Cell-based Assays

Cell-based assays, which are fundamental for understanding of thebiochemical events in cells, have been used with increasing frequency inbiology, pharmacology, toxicology, genetics, and oncology [see, e.g.,Benjamin et al. (1992) Mol. Cell. Biol. 12:2730-2738] Such cell linesmay be constructed or purchased [see, e.g., the Pro-Tox Kit availablefrom Xenometrix, Boulder Colo.; see, also International PCT ApplicationNo. WO 94/7208 cell lines]. Established cell lines, primary cellculture, reporter gene systems in recombinant cells, cells transfectedwith gene of interest, and recombinant mammalian cell lines have beenused to set up cell-based assays. For example Xenometrix, Inc. [Boulder,Colo.] provides kits for screening compounds for toxicological endpointsand metabolic profiles using bacteria and human cell lines. Screening iseffected by assessing activation of regulatory elements of stress genesfused to reporter genes in bacteria, human liver or colon cell lines andprovide information on the cytotoxicity and permeability of testcompounds.

In any drug discovery program, cell-based assays offer a broad range ofpotential targets as well as information on cytotoxicity andpermeability. The ability to test large numbers of compounds quickly andefficiently provides a competitive advantage in pharmaceutical leadidentification.

High throughput screening with cell-based assays is often limited by theneed to use separation, wash, and disruptive processes that compromisethe functional integrity of the cells and performance of the assay.Homogeneous or mix-and-measure type assays simplify investigation ofvarious biochemical events in whole cells and have been developed usingscintillation microplates [see, e.g., International PCT Application No.WO 94/26413, which describes scintillant plates that are adapted forattachment and/or growth of cells and proximity assays using suchcells]. In certain embodiment herein, cell lines such as those describedin International PCT Application No. WO 94/17208 are be plated onscintillant plates, and screened against compounds synthesized onmatrices with memories. Matrices with memories encoded with the identityof the linked molecule will be introduced into the plates, the linkagescleaved and the effects of the compounds assessed. Positive compoundswill be identified by querying the associated memory.

The scintillant base plate is preferably optically transparent toselected wavelengths that allow cells in culture to be viewed using aninverted phase contrast microscope, and permit the material to transmitlight at a given wavelength with maximum efficiency. In addition thebase retains its optical properties even after exposure to incident betaradiation from radioisotopes as well as under stringent radiationconditions required for sterilization of the plates. The base plate canbe composed of any such optically transparent material containingscintillant, e.g., a scintillant glass based on lanthanide metalcompounds. Typically, the base plate is composed of any plasticmaterial, generally formed from monomer units that include phenyl ornaphthyl moieties in order to absorb incident radiation energy fromradionuclides which are in close proximity with the surface. Preferablythe plastic base plate is composed of polystyrene or polyvinyltoluene,into which the scintillant is incorporated. The scintillant includes,but is not limited to: aromatic hydrocarbons such as p-terphenyl,p-quaterphenyl and their derivatives, as well as derivatives of theoxazoles and 1,3,4-oxadiazoles, such as2-(4-t-butylphenyl)-5-(4-biphenyl)-1,3,4-oxadiazole and2,5-diphenyloxazole. Also included in the polymeric composition may be awavelength shifter such as 1,4-bis(5-phenyl-2-oxazolyl)benzene,9,10-diphenylanthracene, 1,4-bis(2-methylstyryl)-benzene, and other suchcompounds. The function of the wavelength shifter is to absorb the lightemitted by the scintillant substance and re-emit longer wavelength lightwhich is a better match to the photo-sensitive detectors used inscintillation counters. Other scintillant substances and polymer bodiescontaining them are known to those of skill in this art [see, e.g.,European Patent Application No. 0 556 005 A1].

The scintillant substances can be incorporated into the plastic materialof the base by a variety of methods. For example, the scintillators maybe dissolved into the monomer mix prior to polymerization, so that theyare distributed evenly throughout the resultant polymer. Alternativelythe scintillant substances may be dissolved in a solution of the polymerand the solvent removed to leave a homogeneous mixture. The base plateof disc may be bonded to the main body of the well or array of wells,which itself may be composed of a plastic material includingpolystyrene, polyvinyltoluene, or other such polymers. In the case ofthe multi-well array, the body of the plate may be made opaque, i.e.,non-transparent and internally reflective, in order to completelyexclude transmission of light and hence minimize “cross-talk.” This isaccomplished by incorporating into the plastic at the polymerizationstage a white dye or pigment, for example, titanium dioxide. Bonding ofthe base plate to the main body of the device can be accomplished by anysuitable bonding technique, for example, heat welding, injection moldingor ultrasonic welding.

For example, a 96-well plate is constructed to the standard dimensionsof 96-well microtiter plates 12.8 cm×8.6 cm×1.45 cm with wells in anarray of 8 rows of 12 wells each. The main body of the plate isconstructed by injection molding of polystyrene containing a loading ofwhite titanium oxide pigment at 12%. At this stage, the wells of themicrotiter plate are cylindrical tubes with no closed end. A base plateis formed by injection molding of polystyrene containing2-(4-t-butylphenyl)-5-(4-biphenyl)-1,3,4-oxadiazole (2%) and9,10-diphenylanthracene (0.5%). The base plate has been silk screenprinted with a grid array to further reduce crosstalk. The base plate isthen fused in a separate operation to the body by ultrasonic welding,such that the grid array overlies the portions of the microtiter platebetween the wells.

A 24-well device is constructed to the dimensions 12.8×8.6×1.4 cm with24 wells in an array of 4 rows of 6 wells. The main body of the plate[not including the base of each well] is constructed by injectionmolding of polystyrene containing 12% white titanium oxide pigment. Thebase 24 of each well is injection molded with polystyrene containing2-(4-t-butylphenyl)-5-(4-biphenylyl)-1,3,4-oxadizaole [2%] and9,10-diphenylanthracene [0.5%]. The heat from the injected base plasticresults in fusion to the main body giving an optically transparent baseto the well.

The plates may contain multiple wells that are continuous or that areeach discontinuous from the other wells in the array, or they may besingle vessels that have, for example, an open top, side walls and anoptically transparent scintillant plastic base sealed around the loweredge of the side walls.

In another format the plate, is a single well or tube. The tube may beconstructed from a hollow cylinder made from optically transparentplastic material and a circular, scintillant containing, plastic disc.The two components are welded together so as to form a single well ortube suitable for growing cells in culture. As in the plate format,bonding of the circular base plate to the cylindrical portion isachieved by any conventional bonding technique, such as ultrasonicwelding. The single well or tube may be any convenient size, suitablefor scintillation counting. In use, the single well may either becounted as an insert in a scintillation vial, or alternatively as aninsert in a scintillation vial, or alternatively as an insert in amulti-well plate of a flat bed scintillation counter. In this lattercase, the main body of the multi-well plate would need to be opaque forreasons given earlier.

The various formats are selected according to use. They may be used forgrowing cells and studying cellular biochemical processes in livingcells or cell fragments. The 96-well plate is a standard format used inexperimental cell biology and one that is suitable for use in a flat bedscintillation counter [e.g., Wallac Microbeta or Packard Top Count]. Inthe multi-well format, it is an advantage to be able to prevent “crosstalk” between different wells of the plate that may be used formonitoring different biological processes using different amounts ortypes of radioisotope. Therefore the main body of the plate can be madefrom opaque plastic material. The 24-well plate format is commonly usedfor cell culture. This type of plate is also suitable for counting in aflat bed scintillation counter. The dimensions of the wells will belarger.

As an alternative format, the transparent, scintillant containingplastic disc is made to be of suitable dimensions so as to fit into thebottom of a counting vessel. The counting vessel is made fromnon-scintillant containing material such as glass or plastic and shouldbe sterile in order to allow cells to grow and the correspondingcellular metabolic processes to continue. Cells are first cultured onthe disc, which is then transferred to the counting vessel for thepurposes of monitoring cellular biochemical processes.

The culture of cells on the scintillation plastic base plate of thewells (or the disc) involves the use of standard cell cultureprocedures, e.g., cells are cultured in a sterile environment at 37° C.in an incubator containing a humidified 95% air/5% CO₂ atmosphere.Various cell culture media may be used including media containingundefined biological fluids such as fetal calf serum, or media which isfully defined and serum-free. For example, MCDB 153 is a selectivemedium for the culture of human keratinocytes [Tsao et al. (1982) J.Cell. Physiol. 110:219-229].

These plates are suitable for use with any adherent cell type that canbe cultured on standard tissue culture plasticware, including culture ofprimary cells, normal and transformed cells derived from recognizedsources species and tissue sources. In addition, cells that have beentransfected with the recombinant genes may also be cultured using theinvention. There are established protocols available for the culture ofmany of these diverse cell types [see, e.g., Freshney et al. (1987)Culture of Animal Cells: A Manual of Basic Technique, 2nd Edition, AlanR. Liss Inc.]. These protocols may require the use of specializedcoatings and selective media to enable cell growth and the expression ofspecialized cellular functions.

The scintillating base plate or disc, like all plastic tissue cultureware, requires surface modification in order to be adapted for theattachment and/or growth of cells. Treatment can involves the use ofhigh voltage plasma discharge, a well established method for creating anegatively charged plastic surface [see, e.g., Amstein et al. (1975) J.Clinical Microbiol. 2:46-54]. Cell attachment, growth and the expressionof specialized functions can be further improved by applying a range ofadditional coatings to the culture surface of the device. These caninclude: (i) positively or negatively charged chemical coatings such aspoly-lysine or other biopolymers [McKeehan et al. (1976) J. Cell Biol.71:727-734 (1976)]; (ii) components of the extracellular matrixincluding collagen, laminin, fibronectin [ see, e.g., Kleinman et al.(1987) Anal. Biochem. 166:1-13]; and (iii) naturally secretedextracellular matrix laid down by cells cultured on the plastic surface[Freshney et al. et al. (1987) Culture of Animal Cells: A Manual ofBasic Technique, 2nd Edition, Alan R. Liss Inc.]. Furthermore, thescintillating base plate may be coated with agents, such as lectins, oradhesion molecules for attachment of cell membranes or cell types thatnormally grow in suspension. Methods for the coating of plasticware withsuch agents are known [see, e.g., Boldt et al. (1979) J. Immunol.123:808].

In addition, the surface of the scintillating layer may be coated withliving or dead cells, cellular material, or other coatings of biologicalrelevance. The interaction of radiolabeled living cells, or otherstructures with this layer can be monitored with time allowing processessuch as binding, movement to or from or through the layer to bemeasured.

Virtually all types of biological molecules can be studied. A anymolecule or complex of molecules that interact with the cell surface orthat can be taken up, transported and metabolized by the cells, can beexamined using real time analysis. Examples of biomolecules will includereceptor ligands, protein and lipid metabolite precursors (e.g., aminoacids, fatty acids), nucleosides and any molecule that can beradiolabeled. This would also include ions such as calcium, potassium,sodium and chloride, that are functionally important in cellularhomeostasis, and which exist as radioactive isotopes. Furthermore,viruses and bacteria and other cell types, which can be radiolabeled asintact moieties, can be examined for their interaction with monolayeradherent cells grown in the scintillant well format.

The type of radioactive isotope that can be used with this system willtypically include any of the group of isotopes that emit electronshaving a mean range up to 2000 μm in aqueous medium. These will includeisotopes commonly used in biochemistry such as [³H], [¹²⁵I], [¹⁴C],[³⁵S], [⁴⁵Ca], [³³p, and [³²p], but does not preclude the use of otherisotopes, such as [⁵⁵Fe], [¹⁰⁹Cd] and [⁵¹Cr] that also emit electronswithin this range. The wide utility of the invention for isotopes ofdifferent emission energy is due to the fact that the current formatsenvisaged would allow changes to the thickness of the layer containing ascintillant substance, thereby ensuring that all the electron energy isabsorbed by the scintillant substance. Furthermore, cross-talkcorrection software is available which can be utilized with all highenergy emitters. Applications using these plates include proteinsynthesis, Ca²⁺ transport, receptor-ligand binding, cell adhesion, sugartransport and metabolism, hormonal stimulation, growth factor regulationand stimulation of motility, thymidine transport, and protein synthesis.

For use in accord with the methods herein, the scintillant plates caninclude a memory in each well, or alternatively, memory withmatrix-linked compounds will be added to each well. The recording devicewith memory may be impregnated or encased or placed in wells of theplate, typically during manufacture. In preferred embodiments, however,the memories are added to the wells with adsorbed or linked molecules.

In one embodiment, matrices with memories with linked molecules areintroduced into scintillant plates in which cells have been cultured[see, e.g., International PCT Application No. WO 94/26413]. For example,cells will be plated on the transparent scintillant base 96-wellmicroplate that permits examination of cells in culture by invertedphase contrast microscope and permits the material to transmit light ata given wavelength with maximum efficiency. Matrices with memories towhich test compounds linked by preferably a photocleaveable linker areadded to the wells. The identity of each test compound is encoded in thememory of the matrix during synthesis if the compound is synthesized onthe matrix with memory or when the compound is linked to the matrix.

Following addition of matrix with memory to the well and release ofchemical entities synthesized on the beads by exposure to light or otherprocedures, the effects of the chemical released from the beads on theselected biochemical events, such as signal transduction, cellproliferation, protein or DNA synthesis, in the cells can be assessed.In this format receptor binding Such events include, but are not limitedto:

whole cell receptor-ligand binding tagonist or antagonist], thymidine oruridine transport, protein synthesis (using, for example, labeledcysteine, methionine, leucine or proline], hormone and growth factorinduced stimulation and motility, and calcium uptake.

In another embodiment, the memories are included in the plates eitherplaced in the plates or manufactured in the wells of the plates. Inthese formats, the identities of the contents of the well is encodedinto the memory. Of course it is understood, that the informationencoded and selection of encased or added memories depends upon theselected protocol.

In another format, cells will be plated on the tissue culture plate,after transferring the matrices with memories and release of compoundssynthesized on the beads in the well. Cytostatic, cytotoxic andproliferative effects of the compounds will be measured usingcolorimetric [MTT, XTT, MTS, Alamar blue, and Sulforhodamine B],fluorimetric [carboxyfluorescein diacetate], or chemiluminescentreagents [i.e., CytoLite™, Packard Instruments, which is used in ahomogeneous luminescent assay for cell proliferation, cell toxicity andmulti-drug resistance].

For example, cells that have been stably or transiently transfected witha specific gene reporter construct containing an inducible promoteroperatively linked to a reporter gene that encodes an indicator proteincan be calorimetrically monitored for promoter induction. Cells will beplated on the tissue culture 96-well microtiter plate and after additionof memories with matrices in the wells and release of chemical entitiessynthesized on the matrices, the effect of the compound released fromthe beads on the gene expression will be assessed. The CytosensorMicrophysiometer [Molecular Devices] evaluates cellular responses thatare mediated by G protein-linked receptors, tyrosine kinase-linkedreceptors, and ligand-gated ion channels. It measures extracellular pHto assess profiles of compounds assessed for the ability to modulateactivities of any of the these cell surface proteins by detectingsecretion of acid metabolites as a result of altered metabolic states,particularly changes in metabolic rate. Receptor activation requires useof ATP and other energy resources of the cell thereby leading toincreased in cellular metabolic rate. For embodiments herein, thememories with matrices, particularly those modified for measuring pH,and including linked test compounds, can be used to track and identifythe added test compound added and also to detect changes in pH, therebyidentifying linked molecules that modulate receptor activities.

3. Memories With Matrices For Non-radioactive Energy Transfer ProximityAssays

Non-radioactive energy transfer reactions, such as FET or FRET, FP andHTRF assays, are homogeneous luminescence assays based on energytransfer are carried out between a donor luminescent label and anacceptor label [see, e.g., Cardullo et al. (1988) Proc. Natl. Acad. Sci.U.S.A. 85:8790-8794; Peerce et al. (1986) Proc. Natl. Acad. Sci. U.S.A.83:8092-8096; U.S. Pat. No. 4,777,128; U.S. Pat. No. 5,162,508; U.S.Pat. No. 4,927,923; U.S. Pat. No. 5,279,943; and International PCTApplication No. WO 92/01225]. The donor label is usually a rare earthmetal cryptate, particularly europium trisbipyridine diamine [EuTBP] orterbium trisbipyridine diamine [TbTBP] and an acceptor luminescent,presently fluorescent, label. When the donor is EuTBP, the acceptor ispreferably allopycocyanin [APC], allophycocyanin B, phycocyanin C orphycocyanin R, and when the donor is TbTBP, the acceptor is a rhodamine,thiomine, phycocyanin R, phycoerythrocyanin, phycoerythrin C,phycoerythrin B or phycoerythrin R.

Energy transfer between such donors and acceptors is highly efficient,giving an amplified signal and thereby improving the precision andsensitivity of the assay. Within distances characteristic ofinteractions between biological molecules, the excitation of afluorescent label (donor) is transferred non radiatively to a secondfluorescent label (acceptor). When using europium cryptate as the donor,APC, a phycobiliprotein of 5 kDa, is presently the preferred acceptorbecause it has high molar absorptivity at the cryptate emissionwavelength providing a high transfer efficiency, emission in a spectralrange in which the cryptate signal is insignificant, emission that isnot quenched by presence of sera, and a high quantum yield. When usingEu³⁺ cryptate as donor, an amplification of emitted fluorescence isobtained by measuring APC emission.

The rare earth cryptates are formed by the inclusion of a luminescencelanthanide ion in the cavity of a macropolycyclic ligand containing2,2′-biphyridine groups as light absorbers [see, e.g., U.S. Pat. No.5,162,508; U.S. Pat. No. 4,927,923; U.S. Pat. No. 5,279,943; andInternational PCT Application No. WO 92/01225]. Preferably the Eu3⁺trisbypryidine diamine derivative, although the acceptor may be used asthe label, is cross-linked to antigens, antibodies, proteins, peptides,and oligonucleotides and other molecules of interest.

For use herein, matrices with memories are prepared that incorporateeither the donor or, preferably the acceptor, into or on the matrix. Inpractice, as with the scintillating matrices with memories, the matricesmay be of any format, i.e. particulate, or continuous, and used in anyassay described above for the scintillating matrices. For example, therecording device is coated with a protective coating, such as glass orpolystyrene. If glass it can be etched. As with preparation of thescintillating matrices with memories, compositions containing the donoror preferably acceptor, such as APC, and typically a polymer or gel, arecoated on the recording device or the device is mixed with thecomposition to produce a fluorescing matrix with memory. To make thesematrices resistant to chemical reaction, if needed, they may be coatedwith polymers such as polyvinylbenzene or polystyrene. Molecules, suchas the constituents of combinatorial libraries, are synthesized on thefluorescing matrices with memories, or molecules or biological particlesare linked thereto, the identity of the synthesized molecules or linkedmolecules or biological particles is encoded in memory, and theresulting matrices with memories employed in any suitable assay,including any of those described for the scintillating memories withmatrices. In particular, these homogeneous assays using long-livedfluorescence rare earth cryptates and amplification by non radiativeenergy transfer have been adapted to use in numerous assays includingassays employing ligand receptor interaction, signal transduction,transcription factors (protein-protein interaction), enzyme substrateassays and DNA hybridization and analysis [see, Nowak (1993) Science270:368; see, also, Velculescu et al. (1995) Science 270:484-487, andSchena et al. (1995) Science 270:467-470, which describe methodsquantitative and simultaneous analysis of a large number of transcriptsthat are particularly suited for modification using matrices withmemories]. Each of these assays may be modified using the fluorescingmatrices with memories provided herein.

For example, a receptor will be labeled with a europium cryptate [wherethe matrices with memories incorporate, for example allophycocyanin(APC)] or will be labeled with APC, where the matrices incorporate aeuropium cryptate. After mixing receptor and mixtures of matrices withdifferent ligands, the mixture is exposed to laser excitation at 337 nm,and, if reaction has occurred, typical signals of europium cryptate andAPC over background are emitted. Measurement with an interference filtercentered at 665 nm selects the signal of the APC labeled receptor fromthat of europium cryptate labeled ligand on the beads. If particulate,the memories of matrices that emit at 665, can be queried to identifylinked ligands.

4. Other Applications Using Memories With Matrices and LuminescingMemories With Matrices a. Natural Product Screening

In the past, the vast majority of mainline pharmaceuticals have beenisolated form natural products such as plants, bacteria, fungus, andmarine microorganisms. Natural products include microbials, botanicals,animal and marine products. Extracts of such sources are screened fordesired activities and products. Selected products include enzymes[e.g., hyaluronidase], industrial chemicals [e.g., petroleum emulsifyingagents], and antibiotics [e.g., penicillin]. It is generally consideredthat a wealth of new agents still exist within the natural productspool. Large mixtures of natural products, even within a fermentationbroth, can be screened using the matrices with memory combinationslinked, for example, to peptides, such as antigens or antibody fragmentsor receptors, of selected and known sequences or specificities, or toother biologically active compounds, such as neurotransmitters, cellsurface receptors, enzymes, or any other identified biological target ofinterest. Mixtures of these peptides linked to memory matrices can beintroduced into the natural product mixture. Individual bindingmatrices, detected by an indicator, such as a fluorometric dye, can beisolated and the memory queried to determine which linked molecule orbiological particle is bound to a natural product.

b. Immunoassays and Immunodiagnostics

The combinations and methods provided herein represent major advances inimmunodiagnostics. Immunoassays [such as ELISAs, RIAs and EIAs (enzymeimmunoassays)] are used to detect and quantify antigens or antibodies.

(1) Immunoassays

Immunoassays detect or quantify very small concentrations of analytes inbiological samples. Many immunoassays use solid supports in whichantigen or antibody is covalently, non-covalently, or otherwise, such asvia a linker, attached to a solid support matrix. The support-boundantigen or antibody is then used as an analyte in the assay. As withnucleic acid analysis, the resulting antibody-antigen complexes or othercomplexes, depending upon the format used, rely on radiolabels or enzymelabels to detect such complexes.

The use of antibodies to detect and/or quantitate reagents [“antigens”]in blood or other body fluids has been widely practiced for many years.Two methods have been most broadly adopted. The first such procedure isthe competitive binding assay, in which conditions of limiting antibodyare established such that only a fraction [usually 30-50%] of a labeled[e.g., radioisotope, fluophore or enzyme] antigen can bind to the amountof antibody in the assay medium. Under those conditions, the addition ofunlabeled antigen [e.g., in a serum sample to be tested] then competeswith the labeled antigen for the limiting antibody binding sites andreduces the amount of labeled antigen that can bind. The degree to whichthe labeled antigen is able to bind is inversely proportional to theamount of unlabeled antigen present. By separating the antibody-boundfrom the unbound labeled antigen and then determining the amount oflabeled reagent present, the amount of unlabeled antigen in the sample[e.g., serum] can be determined.

As an alternative to the competitive binding assay, in the labeledantibody, or “immunometric” assay [also known as “sandwich” assay], anantigen present in the assay fluid is specifically bound to a solidsubstrate and the amount of antigen bound is then detected by a labeledantibody [see, e.g., Miles et al. (1968) Nature 29:186-189; U.S. Pat.No. 3,867,517; U.S. Pat. No. 4,376,110]. Using monoclonal antibodiestwo-site immunometric assays are available [see, e.g., U.S. Pat. No.4,376,110]. The “sandwich” assay has been broadly adopted in clinicalmedicine. With increasing interest in “panels” of diagnostic tests, inwhich a number of different antigens in a fluid are measured, the needto carry out each immunoassay separately becomes a serious limitation ofcurrent quantitative assay technology.

Some semi-quantitative detection systems have been developed [see, e.g.,Buechler et al. (1992) Clin. Chem. 38:1678-1684; and U.S. Pat. No.5,089,391] for use with immunoassays, but no good technologies yet existto carefully quantitate a large number of analytes simultaneously [see,e.g., Ekins et al. (1990) J. Clin. Immunoassay 13:169-181] or to rapidlyand conveniently track, identify and quantitate detected analytes.

The methods and memories with matrices provided herein provide a meansto quantitate a large number of analytes simultaneously and to rapidlyand conveniently track, identify and quantitate detected analytes.

(2) Multianalyte Immunoassays

The combinations of matrix with memories provided herein permits thesimultaneous assay of large numbers of analytes in any format. Ingeneral, the sample that contains an analyte, such as a ligand or anysubstance of interest, to be detected or quantitated, is incubated withand bound to a protein, such as receptor or antibody, or nucleic acid orother molecule to which the analyte of interest binds. In oneembodiment, the protein or nucleic acid or other molecule to which theanalyte of interest binds has been linked to a matrix with memory priorto incubation; in another embodiment, complex of analyte or ligand andprotein, nucleic acid or other molecule to which the analyte of interestbinds is linked to the matrix with memory after the incubation; and in athird embodiment, incubation to form complexes and attachment of thecomplexes to the matrix with memory are simultaneous. In any embodiment,attachment is effected, for example, by direct covalent attachment, bykinetically inert attachment, by noncovalent linkage, or by indirectlinkage, such as through a second binding reaction [i.e., biotin-avidin,Protein A-antibody, antibody-hapten, hybridization to form nucleic acidduplexes of oligonucleotides, and other such reactions andinteractions]. The complexes are detected and quantitated on the solidphase by virtue of a label, such as radiolabel, fluorescent label,luminophore label, enzyme label or any other such label. The informationthat is encoded in the matrix with memory depends upon the selectedembodiment. If, for example, the target molecule, such as the protein orreceptor is bound to the solid phase, prior to complexation, theidentity of the receptor and/or source of the receptor may be encoded inthe memory in the matrix.

For example, the combinations provided herein are particularly suitablefor analyses of multianalytes in a fluid, and particularly formultianalyte immunoassays. In one example, monoclonal antibodies veryspecific for carcinoembryonic antigen [CEA], prostate specific antigen[PSA], CA-125, alphafetoprotein [AFP], TGF-β, IL-2, IL-8 and IL-10 areeach covalently attached to a different batch of matrices with memoriesusing well-established procedures and matrices for solid phase antibodyassays. Each antibody-matrix with memory complex is given a specificidentification tag, as described herein.

A sample of serum from a patient to be screened for the presence orconcentration of these antigens is added to a tube containing two ofeach antibody-matrix with memory complex [a total of 16 beads, orduplicates of each kind of bead]. A mixture of monoclonal antibodies,previously conjugated to fluorescent dyes, such as fluorescein orphenyl-EDTA-Eu chelate, reactive with different epitopes on each of theantigens is then added. The tubes are then sealed and the contents aremixed for sufficient time [typically one hour] to allow any antigenspresent to bind to their specific antibody-matrix with memory-antigencomplex to produce antibody-matrix with memory-antigen-labeled antibodycomplexes. At the end of the time period, these resulting complexes arebriefly rinsed and passed through an apparatus, such as that set forthin FIG. 7, but with an additional light source. As each complex passesthrough a light source, such as a laser emitting at the excitationwavelength of fluorescein, about 494 nm, or 340 nm for the Eu chelatecomplex, its fluorescence is measured and quantitated by reading theemitted photons at about 518 nm for fluorescein or 613 nm forphenyl-EDTA-Eu, and as its identity is determined by the specific signalreceived by the RF detector. In this manner, eight different antigensare simultaneously detected and quantitated in duplicate.

In another embodiment, the electromagnetically tagged matrices withrecorded information regarding linked antibodies can be used with othermultianalyte assays, such as those described by Ekins et al. [(1990) J.Clin. lmmunoassay 13:169-181; see, also International PCT ApplicationsNos. 89/01157 and 93/08472, and U.S. Pat. Nos. 4,745,072, 5,171,695 and5,304,498]. These methods rely on the use of small concentrations ofsensor-antibodies within a few μm² area. Individual memories withmatrices, or an array of memories embedded in a matrix are used.Different antibodies are linked to each memory, which is programmed torecord the identity of the linked antibody. Alternatively, the antibodycan be linked, and its identity or binding sites identified, and theinformation recorded in the memory. Linkage of the antibodies can beeffected by any method known to those of skill in this art, but ispreferably effected using cobalt-iminodiacetate coated memories [see,Hale (1995) Analytical Biochem. 231:46-49, which describes means forimmobilization of antibodies to cobalt-iminodiacetate resin] mediatedlinkage particularly advantageous. Antibodies that are reversibly boundto a cobalt-iminodiacetate resin are attached in exchange insert mannerwhen the cobalt is oxidized from the +2 to +3 state. In this state theantibodies are not removed by metal chelating regents, high salt,detergents or chaotropic agents. They are only removed by reducingagents. In addition, since the metal binding site in antibodies is inthe C-terminus heavy chain, antibodies so-bound are oriented with thecombining site directed away from the resin.

In particular antibodies are linked to the matrices with memories. Thematrices are either in particular form or in the form of a slab with anarray of recording devices linked to the matrices or microtiter dish orthe like with a recording device in each well. Antibodies are thenlinked either to each matrix particle or to discrete “microspots” on theslab or in the microtiter wells. In one application, prior to use ofthese matrices with memories, they are bound to a relatively lowaffinity anti-idiotype antibody [or other species that specificallyrecognizes the antibody binding site, such as a single chain antibody orpeptidomimetic] labeled with a fluophore [e.g., Texas Red, acridine,fluorescein, ellipticine, rhodamine, Lissamine rhodamine B, MalachiteGreen, erythrosin, tetramethylrhodamine, eosin, pyrene, anthracene,methidium, ethydium, phenanthroline, 4-dimethylaminonaphthalene,quinoxaline, 2-dimethylaminonaphthalene,7-dimethylamino-4-methylcoumarin, 7-dimethylaminocoumarin,7-hydroxy-4-methylcoumarin, 7-hydroxycoumarin, 7-methoxycoumarin,7-acetoxycoumarin, 7-diethylamino-3-phenyl-4-methyl-coumarin,isoluminol, benzophenone, dansyl, dabsyl, mansyl, sulfo rhodamine,4-acetamido-4′-stilbene-2,2′-disulfonic acid disodium salt,4-benzamido-4′-stilbene-2,2′-disulfonic acid disodium salt] to measurethe concentration of and number of available binding sites present oneach matrix with memory particle or each microspot, which information isthen encoded into each memory for each microspot or each particle [see,Ekins et al. (1990) J. Clin. Immunoassay 13:169-181]. These low affinityantibodies are then eluted, and the matrices can be dried and storeduntil used.

Alternatively or additionally, the memories in the particles or at eachmicrospot could be programmed with the identity or specificity of thelinked antibody, so that after reaction with the test sample andidentification of complexed antibodies, the presence and concentrationof particular analytes in the sample can be determined. They can be usedfor multianalyte analyses as described above.

After reaction with the test sample, the matrices with memories arereacted with a second antibody, preferably, although not necessarily,labeled with a different label, such as a different fluophore, such asfluorescein. After this incubation, the microspots or each matrixparticle is read by passing the particle through a laser scanner [suchas a confocal microscope, see, e.g., Ekins et al. (1990) J. Clin.Immunoassay 13:169-181; see also U.S. Pat. No. 5,342,633] to determinethe fluorescence intensity. The memories at each spot or linked to eachparticle are queried to determine the total number of available bindingsites, thereby permitting calculation of the ratio of occupied tounoccupied binding sites.

Equilibrium dialysis and modifications thereof has been used to studythe interaction of antibody or receptor or other protein or nucleic acidwith low molecular weight dialyzable molecules that bind to the antibodyor receptor or other protein or nucleic acid. For applications herein,the antibody, receptor, protein or nucleic acid is linked to solidsupport (matrix with memory) and is incubated with the ligand.

In particular, this method may be used for analysis of multiple bindingagents [receptors], linked to matrices with memories, that compete foravailable ligand, which is present in limiting concentration. Afterreaction, the matrices with memories linked to the binding agents[receptors] with the greatest amount of bound ligand, are the bindingagents [receptors] that have the greatest affinity for the ligand.

The use of matrices with memories also permits simultaneousdetermination of K_(a) values of multiple binding agents [receptors] orhave multiple ligands. For example, a low concentration of labeledligand is mixed with a batch of different antibodies bound to matriceswith memories. The mixture is flowed through a reader [i.e., a Coultercounter or other such instrument that reads RF and the label] couldsimultaneously measure the ligand [by virtue of the label] and identityof each linked binding agent for linked ligand] as the chip is read.After the reaction equilibrium [determined by monitoring progress of thereaction] labeled ligand is added and the process of reading label andthe chips repeated. This process is repeated until all binding sites onthe binding agent [or ligand] approach saturation, thereby permittingcalculation of K_(a) values and binding sites that were available.

C. Selection of Antibodies and Other Screening Methods (1) AntibodySelection

In hybridoma preparation and selection, fused cells are plated into, forexample, microtiter wells with the matrices with memory-tagged antibodybinding reagent [such as protein A or Co-chelate [see, e.g., Smith etal. (1992) Methods: A Companion to Methods in Enzymology 4, 73 (1992);III et al. (1993) Biophys J. 64:919; Loetscher et al. (1992) J.Chromatography 595:113-199; U.S. Pat. No. 5,443,816; Hale (1995)Analytical Biochem. 231:46-49]. The solid phase is removed, pooled andprocessed batchwise to identify the cells that produce antibodies thatare the greatest binders [see, e.g., U.S. Pat. No. 5,324,633 for methodsand device for measuring the binding affinity of a receptor to a ligand;or the above method by which phage libraries are screened for highestK_(A) phage, i.e., limiting labeled antigen].

(2) Antibody Panning

Memories with matrices with antibody attached thereto [e.g. particularlyembodiments in which the matrix is a plate] may be used in antibodypanning [see, e.g., Wysocki et al. (1978) Proc. Natl. Acad. Sci. U.S.A.75:2844-48; Basch et al. (1983) J. Immunol. Methods 56:269; Thiele etal. (1986) J. Immunol. 136:1038-1048; Mage et al. (1981) Eur. J.Immunol. 11:226; Mage et al. (1977) J. Immunol. Methods 15:47-56; see,also, U.S. Pat. Nos. 5,217,870 and 5,087,570, for descriptions of thepanning method]. Antibody panning was developed as a means tofractionate lymphocytes on the basis of surface phenotype based on theability of antibody molecules to adsorb onto polystyrene surfaces andretain the ability to bind antigen. Originally [Wysocki et al. (1978)Proc. Natl. Acad. Sci. U.S.A. 75:2844-2848] polystyrene dishes coatedwith antibodies specific for cell surface antigens and permit cells tobind to the dishes, thereby fractionating cells. In embodiments herein,polystyrene or other suitable matrix is associated with a memory deviceand coated with an antibody, whose identity is recorded in the memory.Mixtures of these antibody coated memories with matrices can be mixedwith cells, and multiple cell types can be sorted and identified byquerying the memories to which cells have bound.

d. Phage Display

Phage, viruses, bacteria and other such manipulable hosts and vectors[referred to as biological particles] can be modified to expressselected antigens [peptides or polypeptides] on their surfaces by, forexample, inserting DNA encoding the antigen into the host or vectorgenome, at a site such as in the DNA encoding the coat protein, suchthat upon expression the antigen [peptide or polypeptide] is presentedon the surface of the virus, phage or bacterial host. Libraries of suchparticles that express diverse or families of proteins on their surfaceshave been prepared. The resulting library is then screened with atargeted antigen [receptor or ligand] and those viruses with the highestaffinity for the targeted antigen [receptor or ligand] are selected[see, e.g., U.S. Pat. Nos. 5,403,484, 5,395,750, 5,382,513, 5,316,922,5,288,622, 5,223,409, 5,223,408 and 5,348,867].

Libraries of antibodies expressed on the surfaces of such packages havebeen prepared from spleens of immunized and unimmunized animals and fromhumans. In the embodiment in which a library of phage displayingantibodies from unimmunized human spleens is prepared, it is often ofinterest to screen this library against a large number of differentantigens to identify a number of useful human antibodies for medicalapplications. Phage displaying antibody binding sites derived fromsingle or small numbers of spleen cells can be separately produced,expanded into large batches, and bound to matrices with memories, suchas programmable PROM or EEPROM memories, and identified according tophage batch number recorded in the memory. Each antigen can then beexposed to a large number of different phage-containing memory devices,and those that bind the antigen can be identified by one of severalmeans, including radiolabeled, fluorescent labeled, enzyme labeled oralternate (e.g., mouse) tagged antibody labeled antigen. The encodedinformation in the thus identified phage-containing devices, relates tothe batch of phage reactive with the antigen.

Libraries can also be prepared that contain modified binding sites orsynthetic antibodies. DNA molecules, each encoding proteins containing afamily of similar potential binding domains and a structural signalcalling for the display of the protein on the outer surface of aselected viral or bacterial or other package, such as a bacterial cell,bacterial spore, phage, or virus are introduced into the bacterial host,virus or phage. The protein is expressed and the potential bindingdomain is displayed on the outer surface of the particle. The cells orviruses bearing the binding domains to which target molecules bind areisolated and amplified, and then are characterized. In one embodiment,one or more of these successful binding domains is used as a model forthe design of a new family of potential binding domains, and the processis repeated until a novel binding domain having a desired affinity forthe target molecule is obtained. For example, libraries of de novosynthesized synthetic antibody library containing antibody fragmentsexpressed on the surface have been prepared. DNA encoding syntheticantibodies, which have the structure of antibodies, specifically Fab orFv fragments, and contain randomized binding sequences that maycorrespond in length to hypervariable regions [CDRs] can be insertedinto such vectors and screened with an antigen of choice.

Synthetic binding site libraries can be manipulated and modified for usein combinatorial type approaches in which the heavy and light chainvariable regions are shuffled and exchanged between synthetic antibodiesin order to affect specificities and affinities. This enables theproduction of antibodies that bind to a selected antigen with a selectedaffinity. The approach of constructing synthetic single chain antibodiesis directly applicable to constructing synthetic Fab fragments which canalso be easily displayed and screened. The diversity of the syntheticantibody libraries can be increased by altering the chain lengths of theCDRs and also by incorporating changes in the framework regions that mayaffect antibody affinity. In addition, alternative libraries can begenerated with varying degrees of randomness or diversity by limitingthe amount of degeneracy at certain positions within the CDRs. Thesynthetic binding site can be modified further by varying the chainlengths of the CDRs and adjusting amino acids at defined positions inthe CDRs or the framework region which may affect affinities. Antibodiesidentified from the synthetic antibody library can easily be manipulatedto adjust their affinity and or effector functions. In addition, thesynthetic antibody library is amenable to use in other combinatorialtype approaches. Also, nucleic acid amplification techniques have madeit possible to engineer humanized antibodies and to clone theimmunoglobulin [antibody] repertoire of an immunized mouse from spleencells into phage expression vectors and identify expressed antibodyfragments specific to the antigen used for immunization [see, e.g., U.S.Pat. No. 5,395,750].

The phage or other particles, containing libraries of modified bindingsites, can be prepared in batches and linked to matrices that identifythe DNA that has been inserted into the phage. The matrices are thenmixed and screened with labeled antigen [e.g., fluorescent or enzymatic]or hapten, using an assay carried out with limiting quantities of theantigen, thereby selecting for higher affinity phage. Thus, libraries ofphage linked to matrix particles with memories can be prepared. Thematrices are encoded to identify the batch number of the phage, asublibrary, or to identify a unique sequence of nucleotides or aminoacids in the antibody or antibody fragment expressed on its surface. Thelibrary is then screened with labeled antigens. The antigens are labeledwith enzyme labels or radiolabels or with the antigen bound with asecond binding reagent, such as a second antibody specific for a secondepitope to which a fluorescent antigen binds.

Following identification of antigen bound phage, the matrix particle canbe queried and the identity of the phage or expressed surface protein orpeptide determined. The resulting information represents a profile ofthe sequence that binds to the antigen. This information can be analyzedusing methods known to those of skill in this art.

e. Anti-microbial Assays and Mutagenicity Assays

Compounds are synthesized or linked to matrix with memory. The linkageis preferably a photocleavable linkage or other readily cleavablelinkage. The matrices with memories with linked compounds, whoseidentities are programmed into each memory are the placed on, forexample, 10-cm culture plates, containing different bacteria, fungi, orother microorganism. After release of the test compounds theanti-microbial effects of the chemical will be assessed by looking forlysis or other indicia of anti-microbial activity. In preferredembodiments, arrays of memories with matrices can be introduced intoplates. The memories are encoded with the identity of -the linked orassociated test compound and the position on the array.

The AMES test is the most widely used mutagen/carcinogen screening assay[see, e.g., Ames et al. (1975) Mutation Res. 31:347-364; Ames et al.(1973) Proc. Natl. Acad. Sci. U.S.A. 70:782-786.; Maron et al., (1983)Mutation Research 113:173; Ames (1971) in Chemical Mutagens, Principlesand Methods for their Detection, Vol. 1, Plenum Press, NY, pp 267-282].This test uses several unique strains of Salmonella typhimurium that arehistidine-dependent for growth and that lack the usual DNA repairenzymes. The frequency of normal mutations that render the bacteriaindependent of histidine [i.e., the frequency of spontaneous revertants]is low. The test evaluates the impact of a compound on this revertantfrequency. Because some substances are converted to a mutagen bymetabolic action, the compound to be tested is mixed with the bacteriaon agar plates along with the liver extract. The liver extract serves tomimic metabolic action in an animal. Control plates have only thebacteria and the extract. The mixtures are allowed to incubate. Growthof bacteria is checked by counting colonies. A test is positive wherethe number of colonies on the plates with mixtures containing a testcompound significantly exceeds the number on the corresponding controlplates.

A second type of Ames test [see, International PCT Application No. WO95/10629, which is based on U.S. application Ser. No. 08/011,617; andGee et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:11606-11610;commercially avail from Xenometrix, Boulder Colo.] is of interestherein. This test provides a panel of Salmonella typhimurium strains foruse as a detection system for mutagens that also identifies mutagenicchanges. Although a direct descendant of the traditional Ames Salmonellareverse mutation assay in concept, the Ames II assay provides the meansto rapidly screen for base mutations through the use of a mixture of sixdifferent Salmonella strains.

These new strains carry his mutations listed in the table below. All aredeleted for uvrB and are deficient therefore in excision repair. Inaddition, all six have lipopolysaccharide [rfa] mutations rendering themmore permeable, and all contain the pKM¹⁰¹ plasmid conferring enhancedmutability.

STRAIN BASE CHANGE MUTATION TA7001 A:T → G:C hisG1775 TA7002 T:A → A:ThisC9138 TA7003 T:A → G:C hisG9074 TA7004 G:C → A:T hisG9133 TA7005 G:C→ A:T hisG9130 TA7006 G:C → C:G hisC9070

These strains, which revert at similar spontaneous frequencies[approximately 1 to 10×10⁸] can be exposed and plated separately fordetermining mutational spectra, or mixed and exposed together to assessbroad mutagenic potential. The assay takes 3 days from start to finishand can be performed in 96 well- or 384 well-microtiter plates.Revertant colonies are scored using bromo-creosol purple indicator dyein the growth medium. The mixed strains can be assayed first as part ofa rapid screening program. Since this six strain mixture is slightlyless sensitive than individual strains tested alone, compounds which arenegative for the mix can be retested using all six strains. For all butthe weakest mutagens, the Ames II strain mixture appears to be capableof detecting reversion events even if only one strain is induced torevert. The mixed strains provide a means to perform rapid initialscreening for genotoxins, while the battery of base-specific testerstrains permit mutational spectra analysis.

As modified herein, the test compounds are linked to matrices withmemories, that have been encoded with the identity of the testcompounds. The assays can be performed on multiple test compoundssimultaneously using arrays of matrices with memories or multiplematrices with memories encoded with the identity of the linked testcompound and the array position or plate number into which the compoundis introduced.

f. Hybridization Assays and Reactions (1) Hybridization Reactions

It is often desirable to detect or quantify very small concentrations ofnucleic acids in biological samples. Typically, to perform suchmeasurements, the nucleic acid in the sample [i.e., the target nucleicacid] is hybridized to a detection oligonucleotide. In order to obtain adetectable signal proportional to the concentration of the targetnucleic acid, either the target nucleic acid in the sample or thedetection oligonucleotide is associated with a signal generatingreporter element, such as a radioactive atom, a chromogenic orfluorogenic molecule, or an enzyme [such as alkaline phosphatasel thatcatalyzes a reaction that produces a detectable product. Numerousmethods are available for detecting and quantifying the signal.

Following hybridization of a detection oligonucleotide with a target,the resulting signal-generating hybrid molecules must be separated fromunreacted target and detection oligonucleotides. In order to do so, manyof the commonly used assays immobilize the target nucleic acids ordetection oligonucleotides on solid supports. Presently available solidsupports to which oligonucleotides are linked include nitrocellulose ornylon membranes, activated agarose supports, diazotized cellulosesupports and non-porous polystyrene latex solid microspheres. Linkage toa solid support permits fractionation and subsequent identification ofthe hybridized nucleic acids, since the target nucleic acid may bedirectly captured by oligonucleotides immobilized on solid supports.More frequently, so-called “sandwich” hybridization systems are used.These systems employ a capture oligonucleotide covalently or otherwiseattached to a solid support for capturing detectionoligonucleotide-target nucleic acid adducts formed in solution [see,e.g., , EP 276,302 and Gingeras et al. (1989) Proc. Natl. Acad. Sci. USA86:1173]. Solid supports with linked oligonucleotides are also used inmethods of affinity purification. Following hybridization or affinitypurification, however, if identification of the linked molecule orbiological material is required, the resulting complexes or hybrids orcompounds must be subjected to analyses, such as sequencing. Thecombinations and methods herein eliminate the need for such analyses.

Use of matrices with memories in place of the solid support matricesused in the prior hybridization methods permits rapid identification ofhybridizing molecules. The identity of the linked oligonucleotide iswritten or encoded into the memory. After reaction, hybrids areidentified, such as by radioactivity or separation, and the identify ofhybridizing molecules are determined by querying the memories.

(2) Hybridization Assays

Mixtures nucleic acid probes linked to the matrices with memories can beused for screening in assays that heretofore had to be done with oneprobe at a time or with mixtures of probes followed by sequencing thehybridizing probes. There are numerous examples of such assays [see,e.g., U.S. Pat. No. 5,292,874, “Nucleic acid probes to Staphylococcusaureus” to Milliman, and U.S. Pat. No. 5,232,831, “Nucleic acid probesto Streptococcus Dyopenes” to Milliman, et al.; see, also, U.S. Pat.Nos. 5,216,143, 5,284,747 5,352,579 and 5,374,718]. For example, U.S.Pat. No. 5,232,831 provides probes for the detection of particularStreptococcus species from among related species and methods using theprobes. These probes are based on regions of Streptococcus rRNA that arenot conserved among related Streptococcus species. Particular speciesare identified by hybridizing with mixtures of probes and ascertainingwhich probe(s) hybridize. By virtue of the instant matrices withmemories, following hybridization, the identity of the hybridizingprobes can be determined by querying the memories, and therebyidentifying the hybridizing probe.

9. Combinatorial Libraries and Other Libraries and ScreeningMethododologies

The combinations of matrices with memories are applicable to virtuallyany synthetic scheme and library preparation and screening protocol.These include, those discussed herein, and also methodologies anddevices, such as the Chiron “pin” technology [see, e.g., InternationalPCT application No. WO 94/11388; Geysen et al. (1985) Proc. Natl. Acad.Sci. U.S.A. 82:178; Geysen et al. (1987) J. Immunol. Meth. 102:259-274;Maeji et al. (1994) Reactive Polymers 22:203-212], which relies on asupport composed of annular synthesis components that have an activesurface for synthesis of a modular polymer and an inert support rod thatis positioned axially to the annular synthesis components. This pintechnology was developed for the simultaneous synthesis of multiplepeptides. In particular the peptides are synthesized on polyacrylic acidgrafted on the tip of polyethylene pins, typically arranged in amicrotiter format. Amino acid coupling is effected by immersing the pinsin a microtiter plate. The resulting peptides remain bound to the pinsand can be reused.

As provided herein, “pins” may be linked to a memory or recordingdevice, preferably encasing the device, or each pin may be coded and thecode and the identity of the associated linked molecule(s) stored in aremote memory. As a result it will not be necessary to physically arraythe pins, rather the pins can be removed and mixed or sorted.

Also of interest herein, are DIVERSOMER™ technology libraries producedby simultaneous parallel synthesis schemes for production ofnonoligomeric chemical diversity [see, e.g., U.S. Pat. No. 5,424,483;Hobbs DeWitt et al. (1994) Drug Devel. Res. 33:116-124; Czarnik et al.(1994) Polym. Prepr. 35:985; Stankovic et al. (1994) in InnovationPerspect. Solid Phase Synth. Collect. Pap., Int. Symp., 3rd Epton, R.(Ed), pp. 391-6; DeWitt et al. (1994) Drug Dev. Res. 33:116-124; HobbsDeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909-6913]. Inthis technology a starting material is bonded to a solid phase, such asa matrix material, and is subsequently treated with reagents in astepwise fashion. Because the products are linked to the solid support,multistep syntheses can be automated and multiple reactions can beperformed simultaneously to produce libraries of small molecules. Thistechnology can be readily improved by combining the matrices withmemories or encoding the matrix supports in accord with the methodsherein.

The matrices with memories, either those with memories in proximity orthose in which the matrix includes a code stored in a remote memory, canbe used in virtually any combinatorial library protocol. These protocolsor methodologies and libraries, include but are not limited to thosedescribed in any of following references: Zuckermann et al. (1994) J.Med. Chem. 37:2678; Martin et al. (1995) J. Med. Chem. 38:1431; Campbellet al. (1995) J. Am. Chem. Soc. 117:5381; Salmon et al. (1993) Proc.Natl. Acad. Sci. U.S.A. 90:11708; Patek et al. (1994) Tetrahedron Lett.35:9169; Patek et al. (1995) Tetrahedron Lett. 36:2227; Hobbs DeWitt etal. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6906; Baldwin et al. (1995)J. Am. Chem. Soc. 117:5588; and others.

h. Nucleic Acid Sequencing

Methods of DNA sequencing based on hybridization of DNA fragments with acomplete set of fixed length oligonucleotides [usually 8-mers] that areimmobilized individually as dots in a 2-dimensional matrix is sufficientfor computer-assisted reconstruction of the sequences of fragments up to200 bases long [International PCT Application WO 92/10588]. The nucleicacid probes are of a length shorter than a target, which is hybridizedto the probes under conditions such that only those probes having anexact complementary sequence are hybridized maximally, but those withmismatches in specific locations hybridize with a reduced affinity, ascan be determined by conditions necessary to dissociate the pairs ofhybrids. Alignment of overlapping sequences from the hybridizing probesreconstructs the complement of the target [see, EP 0 535 242 Al,International PCT Application WO 95/00530, and Khrapko et al. (1989)FEBS Lttrs. 256:118-122]. The target fragment with the sequence ofinterest is hybridized, generally under highly stringent conditions thattolerate no mismatches or as described below a selected number ofmismatches, with mixtures of oligonucleotides [typically a mixture ofoctomers of all possible sequences] that are each immobilized on amatrix with memory that is encoded with the sequence of the probe. Uponhybridization, hybridizing probes are identified by routine methods,such as OD or using labeled probe, and the sequences of the hybridizingprobes can be determined by retrieving the sequences from the linkedmemories. When hybridization is carried out under conditions in which nomismatches are tolerated, the sequence of the target can then bedetermined by aligning overlapping sequences of the hybridizing probes.

Previous methods used to accomplish this process have incorporatedmicroscopic arrays of nucleotide oligomers synthesized on small siliconbased chips. It is difficult to synthesize such arrays and qualitycontrol the large number of spots on each chip [about 64,000 spots for8-mer oligonucleotides, that number necessary to accomplish sequencingby hybridization].

In the present method, each oligomer is independently synthesized on abatch of individual chips, those chips are tested for accuracy andpurity of their respective oligomers, then one chip from each batch isadded to a large pool containing oligomers having all possiblesequences. After hybridization in batch mode with the gene segment to besequenced, usually amplified by a method such as PCR, using appropriateprimers, and labeled with a detectable [such as fluorescent] tag, thechips can be passed through a detector, such as described above forprocessing multiplexed assays,including multiplexed immunoassays, andthe degree of binding to each oligomer can be determined. After exposingthe batch to varying degrees of dissociating conditions, the devices canagain be assayed for degree of binding, and the strength of binding torelated sequences will relate the sequence of the gene segment [see,e.g., International PCT Application WO 95/00530].

An exemplary method for synthesizing an oligonucleotide library,preferably hexamers or octomers, for use in sequencing methods, or othermethods, is set forth in the EXAMPLES and depicted in FIG. 33, in whichthe oligonucleotides are synthesized on optical memory devices that areeach uniquely encoded either before, during or after synthesis with acode. The identity of the oligomer associated with each code is storedin a remote memory, generally a computer. Other memory with matrices,such as the MICROTUBES™ and MICROKANS™ and other such combination, maybe used in place of the optical memory devices

In particular this library of microreactors [oligonucleotides-linked tomemory with matrix] can be used in methods for DNA sequencing by primerwalking using capillary electrophoresis (CE) and ultrathin slab gels forseparation [see, e.g., Ruiz-Martinez et al. (1996) Biotechniques20:1058-1069; Kieleczawa et al. (1992) Science 258:1787-1791; McCombieet al. (1994) Biotechniques 17:574-5790]. Such methods rely on the useof oligonucleotide libraries, containing all permutations of pentamer orhexamers, which are used as primers. The identity of eacholigonucleotide will be encoded in the associated memory or stored inthe proximate or linked memory. A synthetic protocol is depicted in FIG.33.

i. Separations, Physical Mapping and Measurements of Kinetics of Bindingand Binding Affinities

Multiple blots [i.e., Western, Northern, Southern and/or dot blots] maybe simultaneously reacted and processed. Each memory, in the form of arectangle or other suitable, is linked or coated on one surface withmaterial, such as nitrocellulose, to which or the analyte of interestbinds or with which it reacts. The chips are arranged in an array, suchas in strips that can be formed into rectangles or suitable othershapes, circles, or in other geometries, and the respective x-ycoordinate or other position-identifying coordinates, and, if needed,sheet number and/or other identifying information, is programmed intoeach memory. Alternatively, they may be programmed with thisidentification, then positioned robotically or manually into an arrayconfiguration. They are preferably linked together, such as byreversible glue, or placing them in agarose, or by any suitable methodas long as the reactive surface is not disturbed. Following transfer ofthe material, such as transfer of protein from a Western Blot, nucleicacid from a Southern or Northern blot, dot blots, replica platedbacterial culture, or viral plaques, the memories are separated andmixed for reaction with a traditionally labeled, such as a fluorescentlabel, detection nucleic acid, protein, antibody or receptor ofinterest. Complexes are identified, and their origin in the blotdetermined by retrieving the stored information in each chip.Quantitation may also be effected based on the amount of label bound.

A series of appropriately activated matrices with memories are arrangedin an array, one or, preferably two dimensional. In one configuration,each chip is pre-programmed and placed in a specific location that isentered into its memory, such as an x-y coordinate. At least one surfaceof the memory with matrix is treated so that the transferred reagentbinds. For example, a piece of nitrocellulose can be fixed to one sideof the memory device. The resulting array is then contacted with aseparation medium whereby each reagent of interest is transferred to andbound to the end of the matrix with memory such that the reagentlocation is known. The matrices are separated and pooled; multiplearrays may be pooled as long as source information is recorded in eachmemory. All matrices with memories are then contacted with detectionagents that specifically bind to reagents in the mixture. The matriceswith memories are passed through a reading device, either after anincubation for end point determinations or continuously for kineticmeasurements. The reading devices is a device that can detect label,such as fluorescence, and an reader, such as an RF ready, that can querythe memory and identify each matrix. The rate of binding and maximumbinding and identify of bound reagents can be determined. Dot blots, forexample, can be used in hybridoma analysis to identify clones thatsecrete antibodies of desired reactivity and to determine the relativeaffinities of antibodies secreted by different cell lines. Matrices withmemories that are activated to bind immunoglobulins and with on-boardinformation specifying their relative locations in the array are dippedin an array into the wells of microplates containing hybridoma cells.After incubation, they are withdrawn, rinsed, removed and exposed tolabeled antigen. Matrices of desired specificity and affinity areselected and read thereby identifying the original wells containing thehybridoma cells that produce the selected antibodies.

In other embodiments, the transfer medium [i.e., the nitrocellulose orother such medium] may be part of the surface of the chip or array ofchips that can bind to the separated species subsequent to separation.For example, the separation system, such as the agarose orpolyacrylamide gel, can be included on the surface(s) of the matrix withmemories in the array. After separation the surface will be activatedwith a photoactivatable linker or suitable activating agent to therebycovalently link, such as by a photoflash, the separated molecules to thematrices in the array.

Alternatively, each matrix with memory may have one or more specificbinding agents, such as an antibody or nucleic acid probe, attached(adsorbed, absorbed, or otherwise in physical contact) to matrix withmemory. The matrix with memory and linked binding agent is thencontacted with a medium containing the target(s). After contacting,which permits binding of any targets to which the linked binding agentsspecifically bind, the matrix with memory is processed to identifymemories with matrices to which target has specifically bound viainteraction with the binding agent. For example, the (1) the target islabeled, thereby permitted direct detection of complexes; (2) the memorywith matrix is then contacted with a developing agent, such as a secondantibody or detection probe, whereby binding agent-target complexes aredetected; or (3) the detection agent is present during the reaction,such as non-specifically attached to the matrix with memory or by othermethod [thin film, coated on the matrix with memory, coated onnitrocellulose].

Such support bound analytes may also be used to analyze the kinetics ofbinding by continuously passing the supports through a label readingdevice during the reaction, and identify the labeled complexes. Thebinding agents can be eluted, either in a kinetically readable manner orin batch. In addition, since the recording devices may also includecomponents that record reaction conditions, such as temperature and pH,kinetics, which are temperature and pH dependent, may be accuratelycalculated.

After elution, the support bound analytes may be identified to analyzekinetics of binding to the binding agent. Such binding and elutionprotocols may also be adapted to affinity purification methodologies.

j. Cell Sorting

The devices herein may also be used in methods of cell sorting. Forexample, the memory with matrix combinations are linked to selectedantigens, information regarding the antigens is encoded into thememories, the resulting combinations are used in multi-analyte analysesof cells.

It is possible to identify a profile of cells exhibiting differentsurface markers [antigens, for example, or other ligands or receptormolecules] by using combinations of labeled and matrix memory-boundbinding agents. In one embodiment, each agent, such as an antibody,capable of binding specifically to one of many different surface markersis bound to a different matrix with a memory. The nature of therecognized marker is recorded in the memory of each matrix-binding agentcomplex, and the mixture of binding-agent-matrix memory complexes isreacted with a mixture of cells. The cell-matrix complexes that resultfrom binding agents attaching cells to the surfaces of the respectivematrices are then reacted with a labeled [for example, fluorescent]reagent or mixture of reagents which also reacts with the cells. Theselabeled reagents can be the same or different from those coupled to thememory matrices. When the matrices are passed through a reader [to readthe label and the memory], those that have bound cells can be identifiedand if necessary isolated. This application is particularly useful forscreening for rare cells, for example stem cells in a bone marrow orperipheral lymphocyte sample, for detecting tumor cells in a bone marrowsample to be used for autologous transplantation, or for fetal cells ina maternal circulation.

In these embodiments, the memory with matrices herein can be counted andread with instruments, such as a device that operates on the principlesof a Coulter counter, that are designed to count cells or particles. Inusing a Coulter Counter, a suspension of cells or particles is suckedthrough a minute hole in a glass tube. One electrode is placed withinthe tube and another is outside of the tube in the suspension. Thepassage of a particle through the hole temporarily interrupts thecurrent; the number of interruptions is determined by a conventionalscaling unit.

For use herein, such instruments are modified by including an RF reader[or other reader if another frequency or memory means is selected] sothat the identity of the particle or cell [or antigen on the cell orother encoded information) can be determined as the particle or cellpasses through the hole and interrupts the current, and also, if needed,a means to detect label, such as fluorescent label. As the particlepasses through the hole the RF reader will read the memory in the matrixthat is linked to the particle. The particles also may be countedconcurrently with the determination of the identity of the particle.Among the applications of this device and method, is a means to sortmultiple types of cells at once.

k. Multiplexed or Coupled Protocols in Which the Synthesis Steps [theChemist] is Coupled to Subsequent Uses of the Synthesized Molecules

Multiplexed or multiple step processes in which compounds aresynthesized and then assayed without any intermediate identificationsteps are provided herein. Since the memories with matrices permitidentification of linked or proximate or associated molecules orbiological particles, there is no need to identify such molecules orbiological particles during any preparative and subsequent assayingsteps or processing steps. Thus, the chemistry [synthesis] can bedirectly coupled to the biology [assaying, screening or any otherapplication disclosed herein]. For purposes herein this coupling isreferred to as multiplexing. Thus, high speed synthesis can be coupledto high throughput screening protocols.

H. Applications of the Memories With Matrices and Luminescing MatricesWith Memories in Combinatorial Syntheses and Preparation of Libraries

Libraries of diverse molecules are critical for identification of newpharmaceuticals. A diversity library has three components: solid supportmatrix, linker and synthetic target. The support is a matrix material asdescribed herein that is stable to a wide range of reaction conditionsand solvents; the linker is selectively cleavable and does not leave afunctionalized appendage on the synthetic target; and the target issynthesized in high yield and purity. For use herein, the diversitylibrary further includes a memory or recording device in combinationwith the support matrix. The memory is linked, encased, in proximitywith or otherwise associate with each matrix particle, whereby theidentify of synthesized targets is written into the memory.

The matrices with memories are linked to molecules and particles thatare components of libraries to electronically tagged combinatoriallibraries.

Particularly preferred libraries are the combinatorial libraries thatcontaining matrices with memories that employ radio frequencies forreading and writing.

1. Oligomer and Polypeptide Libraries a. Bio-oligomer Libraries

One exemplary method for generating a library [see, U.S. Pat. No.5,382,5131 involves repeating the steps of (1) providing at least twoaliquots of a solid phase support; separately introducing a set ofsubunits to the aliquots of the solid phase support; completely couplingthe subunit to substantially all sites of the solid phase support toform a solid phase support/new subunit combination, assessing thecompleteness of coupling and if necessary, forcing the reaction tocompleteness; thoroughly mixing the aliquots of solid phase support/newsubunit combination; and, after repeating the foregoing steps thedesired number of times, removing protecting groups such that thebio-oligomer remains linked to the solid phase support. In oneembodiment, the subunit may be an amino acid, and the bio-oligomer maybe a peptide. In another embodiment, the subunit may be a nucleoside andthe bio-oligomer may be an oligonucleotide. In a further embodiment, thenucleoside is deoxyribonucleic acid; in yet another embodiment, thenucleoside is ribonucleic acid. In a further embodiment, the subunit maybe an amino acid, oligosaccharide, oligo-glycosides or a nucleoside, andthe bio-oligomer may be a peptide-oligonucleo-tide chimera or otherchimera. Each solid phase support is attached to a single bio-oligomerspecies and all possible combinations of monomer [or multimers incertain embodiments] subunits of which the bio-oligomers are composedare included in the collection.

In practicing this method herein, the support matrix has a recordingdevice with programmable memory, encased, linked or otherwise attachedto the matrix material, and at each step in the synthesis the supportmatrix to which the nascent polymer is attached is programmed to recordthe identity of the subunit that is added. At the completion ofsynthesis of each biopolymer, the resulting biopolymers linked to thesupports are mixed.

After mixing an acceptor molecule or substrate molecule of interest isadded. The acceptor molecule is one that recognizes and binds to one ormore solid phase matrices with memory/bio-oligomer species within themixture or the substrate molecule will undergo a chemical reactioncatalyzed by one or more solid phase matrix with memory/bio-oligomerspecies within the library.

The resulting combinations that bind to the acceptor molecule orcatalyze reaction are selected. The memory in the matrix-memorycombination is read and the identity of the active bio-oligomer speciesis determined.

b. Split Bead Sequential Syntheses

Various schemes for split bead syntheses of polymers [FIG. 1], peptides[FIG. 2], nucleic acids [FIG. 3] and organic molecules based on apharmacophore monomer [FIG. 4] are provided. Selected matrices withmemory particles are placed in a suitable separation system, such as afunnel [see, FIG. 5]. After each synthetic step, each particle isscanned [i.e., read] as it passes the RF transmitter, and informationidentifying the added component or class of components is stored inmemory. For each type of synthesis a code can be programmed [i.e., a 1at position 1,1 in the memory could, for example, represent alanine atthe first position in the peptide]. A host computer or decoder/encoderis programmed to send the appropriate signal to a transmitter thatresults in the appropriate information stored in the memory [i.e, foralanine as amino acid 1, a 1 stored at position 1,1]. When read, thehost computer or decoder/encoder can interpret the signal read from andtransmitted from the memory.

In an exemplary embodiment, a selected number of beads [i.e.,particulate matrices with memories [matrix particles linked to recordingdevices], typically at least 10³, more often 10⁴, and desirably at least10⁵ or more up to and perhaps exceeding 10¹⁵, are selected or prepared.The beads are then divided into groups, depending upon the number ofchoices for the first component of the molecule. They are divided into anumber of containers equal to or less than [for pooled screening, nestedlibraries or the other such methods] the number of choices. Thecontainers can be microtiter wells, Merrifield synthesis vessels,columns, test tubes, gels, etc. The appropriate reagents and monomer areadded to each container and the beads in the first container are scannedwith electromagnetic with radiation, preferably high frequency radiowaves, to transmit information and encode the memory to identify thefirst monomer. The beads in the second container are so treated. Thebeads are then combined and separated according to the combinatorialprotocol, and at each stage of added monomer each separate group islabeled by inputting data specific to the monomer. At the end of thesynthesis protocol each bead has an oligomer attached and informationidentifying the oligomer stored in memory in a form that can beretrieved and decoded to reveal the identity of each oligomer.

An 8-member decapeptide library was designed, synthesized, and screenedagainst an antibody specifically generated against one of the librarymembers using the matrices with memories. Rapid and clean encoding anddecoding of structural information using radio frequency signals,coupling of combinatorial chemical synthesis to biological assayprotocols, and potential to sense and measure biodata using suitablebiosensors, such as a temperature thermistor or pH electrode, embeddedwithin the devices have been demonstrated. The “split and pool” method[see, e.g., Furka et al. (19910 Int. J. Pent. Protein Res. 37:487-493;Lam et al. (1991) Nature 354:82-84; and Sebestyén et al. (1993) Bioorg.Med. Chem. Lett. 3:413-418] was used to generate the library. An ELISA[see e.g., Harlow et al. (1988) Antibodies, a laboratory manual, ColdSpring Harbor, N.Y.] was used to screen the library for the peptidespecific for the antibody.

2. “Nested” Combinatorial Library Protocols

In this type of protocol libraries of sublibraries are screened, and asublibrary selected for further screening [see, e.g., Zuckermann et al.(1994) J. Med. Chem. 37:2678-2685; and Zuckermann et al. (1992) J. Am.Chem. Soc. 114:10646-10647]. In this method, three sets of monomers werechosen from commercially available monomers, a set of four aromatichydrophobic monomers, a set of three hydroxylic monomers, a set ofseventeen diverse monomers, and three N-termini were selected. Theselection was based on an analysis of the target receptor and knownligands. A library containing eighteen mixtures, generated from the sixpermutations of the three monomer sets, times three N-termini wasprepared. Each mixture of all combinations of the three sets of amines,four sets of hydrophobic monomers and seventeen diverse monomers wasthen assayed. The most potent mixture was selected for deconvolution bysynthesis of pools of combinatorial mixtures of the components of theselected pool. This process was repeated, until individual compoundswere selected.

Tagging the mixtures with the matrices with memories will greatlysimplify the above protocol. instead of screening each mixtureseparately, each matrix particle with memory will be prepared with setsof the compounds, analogous to the mixtures of compounds. The resultingmatrix particles with memories and linked compounds can be combined andthen assayed. As with any of the methods provided herein, the linkedcompounds [molecules or biological particles] can be cleaved from thematrix with memory prior to assaying or anytime thereafter, as long asthe cleaved molecules remain in proximity to the device or in somemanner can be identified as the molecules or particles that were linkedto the device. The matrix particle(s) with memories that exhibit thehighest affinity [bind the greatest amount of sample at equilibrium] areselected and identified by querying the memory to identify the group ofcompounds. This group of compounds is then deconvoluted and furtherscreened by repeating this process, on or off the matrices withmemories, until high affinity compounds are selected.

3. Other Combinatorial Protocols

The matrices with memories provided herein may be used as supports inany synthetic scheme and for any protocol, including protocols forsynthesis of solid state materials. Combinatorial approaches have beendeveloped for parallel synthesis of libraries of solid state materials[see, e.g., Xiang et al. (1995) Science 268:1738-1740]. In particular,arrays containing different combinations, stoichiometries, anddeposition sequences of inorganics, such as BaCO₃, BiO₃, CaO, CuO, PbO,SrCO₃ and Y₂O₃, for screening as superconductors have been prepared.These arrays may be combined with memories that identify position andthe array and/or deposited material.

I. Microvessel Opening and Closing Devices

In order to facilitate the opening and closing of a MICROKAN microvesselor other such microvessel or microreactor, a pair of hand tools isprovided herein. More specifically, and with reference to U.S. Pat. Nos.4,651,598 and 4,662,252, each of the hand tools includes a pliers bodythat has been adapted to accept various portions of the microvessel.Referring to FIG. 44, the cap sealing tool is shown and generallydesignated 4600. As shown, the tool 4600 includes a pair of elongatedhandle portions 4602 and 4604. These handle portions articulate about asliding pivot disc 4605 that engages any one of the teeth 4607 such thatwhen the handles are forced together, the opposite ends of the handleportions also move towards each other. On the ends of the elongatedhandle members, the traditional pliers are modified to have a strikingplate 4610 and a receiving cylinder. The receiving cylinder is pivotallyattached to the end of member 4602 such that the cylinder may swing outaway from the pliers, to facilitate loading and unloading the cylinder4608. On the upper member 4612, the striking plate 4610 is attached tobe positioned directly above the cylinder when the cylinder is in itsraised position. FIG. 45 is a front cross-sectional view showing theplacement of the microvessel within the cylinder such that the lid 4620is positioned over the tube 4616. Referring to FIG. 46, the strikingplate 4610 and cylinder 4622 are shown in lateral cross-section. Fromthis view it is clear that the inside of the cylinder 4622 is formed toaccept a MICROKAN microvessel. As can be appreciated, however, thecylinder may be formed to accept virtually a container of any geometryor size, preferably a container with a volume of 1 ml or less, that issealed as provided herein. The MICROKAN microvessel shown here containsa tag 4618, such as, for exemplification purposes, an RF tag in theshape of a capsule, such as that available from IDTag, describedelsewhere herein. As with the microvessel, the tags may be formed in avariety of shapes and sizes, as long as it is insertable into thecontainer. FIG. 45 shows the lid 4620 of the tube 4616 positioned abovethe tube such that when the handle portions are squeezed together, thelid 4620 is forced into the container to seal the tag therein.

Moving now to FIG. 47, the cylinder 4622 is shown forced against thestriking plate 4610 to press the lid 4614 into the microvessel 4616.Once the tag is contained within the microvessel, the pliers are openedand the cylinder is articulated outwards about the pinion 4626 indirection 4628 such that the tang 4626 strikes the bottom 4630 of themicrovessel 4616 to push the microvessel out of the cylinder. Once thecylinder is articulated, the microvessel may be easily removed from thecylinder. As can be seen from this view, the tag 4618 is sealed insidethe microvessel to prevent exposure of the tag to environmentalcontaminates. Moreover, by placing the tag in a sealable container, thetag may be reused.

Because the tag 4618 may be reused, another tool has been created tofacilitate removing it from the microvessel. Referring now to FIG. 49, asimilar plier-like tool is formed with a striking plate 4610, and a fork4634. The fork is attached to the end of elongated handle 4604 and hastwo prongs 4632 which are connected to define an arc shaped wedge. Thisarc shaped wedge can be positioned against the side of the microvesselwhere the lid 4614 joins the microvessel, and upon actuating the pliers,the prongs slide between the lid and the microvessel to remove the lid.

As perhaps more clearly shown in FIG. 50, the prongs 4632 are wedgeshaped such that the more the pliers are closed, the more the lid isurged out of the microvessel. From this view, the shape of the strikingplate 4610 can be easily seen. It is to be appreciated that any numberof striking plate shapes could be used. In fact, because a container ofany shape could be used to hold the tag, it should be appreciated thatthe striking plate could be shaped to accommodate those shapes. Further,the prongs 4632 on the fork 4634 could be shaped to more closely fit analternatively shaped container. Once the lid is removed, the tag canalso be removed from the microvessel and reused in another microvessel.

J. Sleeves With Memories

In another embodiment provided herein, depicted in FIGS. 35-43, asleeve, containing a remotely programmable memory with a coiled antenna,that is specially adapted to fit on a tube, such as an Hewlett PackardHPLC tube. The sleeve fits tightly on the tube, thereby permitting thetube to be tracked and information about the contents, source or othersome information, to be stored in the memory in the sleeve or in aremote computer that associates the memory with such information.

Referring to FIG. 35, the identification system is shown and generallydesignated 3000. The system 3000 includes a read/write controller 3002that includes a housing 3018 that supports a carousel 3006, and acomputer system 3004. The carousel 3006 as shown is mounted to rest onthe top surface 3020 of the housing 3018. As will be further discussedbelow in connection with FIG. 36, the carousel rotates about itsvertical axis so that as it rotates, each of the ports 3008 will rotatein front of the plunger 3024. The carousel is formed with a number ofthe ports 3008 that are sized to receive a vial 3010. It is to beappreciated that although the carousel is shown to contain 21 differentports, a carousel having any number of ports could be formed.

Formed in the carousel is a keyway 3012 that is sized to accept a key3014. While not shown in this Figure. this key is attached to a hub 3056(shown in FIG. 37). In an effort to stop the carousel from freelyrotating, the plunger 3024 is positioned on the top surface 3020 so thatits slide 3044 (shown in FIG. 36) strikes the outside rim of thecarousel.

The computer system 3004 includes a central processing unit 3030 thathas a serial port to accept a serial cable 3038, a monitor 3032 having ascreen 3034, and a keyboard 3036. While a traditional computer is shownto include separate parts, a laptop computer would be equally acceptableso long as it is equipped with a serial port to accept cable 3038.

Referring now to FIG. 36, the read/write controller is shown from thetop thereby providing a more detailed view of the rotational positioningof the carousel 3006 and the plunger 3024. The plunger is attached tothe top surface 3020 of the housing 3018 with a flange 3042. Thepositioning of the plunger is important because the plunger slide 3044must hit the outer rim of the carousel in a location that will provide astopping pressure against the carousel., This stopping pressure acts toprevent any spinning of the carousel unless there is a turning forceplaced on the carousel itself. It will be appreciated that the strike ofthe plunger could be replaced by a more precise stopping member, such asa post. The post, like the slide, would strike the outer surface of thecarousel. If the post is significantly smaller than the slide, thecarousel may be formed with a number of holes spaced along the rim ofthe carousel so that when activated, the plunger would urge the postinto the hole to securely stop the carousel from spinning. In addition,any other method of orienting the carousel on the housing could be used,so long as the carousel could be rotated.

The surface 3016 of housing 3018 is attached using screws 3022.

Instead of using screws, virtually any manner of attaching the housingtogether may be used. The housing 3018 may be formed from one singlepiece of material that is either bored or machined to have a hollowinside for holding the necessary electronics discussed below.

Also from FIG. 36, the interaction between the key 3014 and keyway 3012is clearly shown. From this view, it is to be appreciated that thecarousel could simply be lifted off of the housing and keyway andreplaced with another carousel that was oriented such that the keyway inthe new carousel would match the position of the key on the housing. Insuch a manner, virtually any number of carousals could be placed on thehousing.

The outer surface of the carousel is formed with 21 ports 3008. Each ofthese ports is formed with a cutout 3009. As easily appreciated fromthis view, the carousel could be made to have a larger diameter toaccommodate a larger number of vials, or the vials could be smaller toaccomplish the same quantity of vials. In other words, by making thecarousel and vials of differing sizes, a virtually unlimited number ofvials could be accommodated. Moreover, because the carousals areremovable, a large number of vials could be processed through theread/write controller in a short period of time. It should also beappreciated that although the carousel is formed with a handle 3026, itcould instead be formed with a grip that would be easily mated with someautomated handling device, such as a robotic arm (not shown). Thisrobotic arm could be independently operated, or could be controlled bythe computer system 3004.

There is a notch 3040 formed in the outer edge of the carousel that isparticularly useful for aligning with the plunger 3024 when installingor removing the carousel from the read/write controller. As is to beappreciated, when the carousel is rotated such that the notch isadjacent the plunger 3024, the slide 3044 will not strike the carousel.As a result, the carousel may be easily lifted up and off of theread/write controller. Such simple removal is particularly useful whenthere are a large number of vials present on the carousel that would adda significant weight to the carousel, making removal more strenuous.

Referring now to FIG. 37, the housing 3018, carousel 3006, vial 3010,and plunger 3024 are shown in cross-section. The housing is shown havinga module 3068 that includes the majority of the electronics needed tooperate the system 3000, with the exception of the computer system 3004.Mounted and extending through the housing 3018 is a motor 3052. Themotor is positioned to extend vertically upwards from the housing andinto a hub 3056 that is sized to receive the carousel 3006.Specifically, the shaft 3054 of the motor extends upwards and is formedwith a locking tab 3058 that is intended to keep the hub properlypositioned on the shaft. In other words, the tab 3058 engages the hub toprevent the hub from rotating when the shaft 3054 is not rotating. Toassist the shaft in maintaining a proper vertical alignment, a bearing3082 is mounted to the housing 3018. Any type of bearing could be usedfor this application, including but not limited to ball bearings orroller bearings. In fact, a grease bearing, if providing sufficientstability, could be used. In the event that the carousel is to bemanually rotated, there would be no need for the motor 3052 and, as aresult, the bearing 3082 would be of a different type to give support tothe carousel itself. In such an instance, the shaft would be fixed tothe housing, or to a bearing mounted on the housing, and the carouselwould rotate freely about the shaft. Thus, in such an instance where nomotor is used, the tabs 3058 would not be formed on the shaft 3054.

In order to control the motor 3052, if used, a control wire 3074 extendsfrom the motor to the module 3068. One function of this module would beto receive an electronic signal from the computer system and command themotor to a particular rotational position. In order to achieve thispositional accuracy, the motor may be a stepper motor, or may beequipped with either a synchro or resolver that would be used todetermine the angular position of the carousel 3006. Briefly, and as isgenerally known in the art, a synchro or resolver would provide athree-phase electrical signal that would represent the angular positionof the carousel. This three-phase signal can be decoded using a synchroor resolver-to-digital converter to determine the angular position ofthe carousel in digital representation. Alternatively, the carouselcould be formed with angular markings on the outer surface of thecarousel that could be read by either a mechanical or optical devicecommon for use in such positioning systems. Such an optical system coulduse a decoding scheme based on a binary-coded-decimal representation ina bar-code form that could be easily marked on the outer surface of thecarousel.

Plunger 3024 is shown with slide 3044 striking the outer surface of vial3010 to hold the rotational position of the carousel. As shown, theplunger 3024 has a slide 3044 that is urged towards the carousel by aspring 3046. As a result of the expansion of the spring, the slide isurged gently against the surface of the carousel. Thus, in order toadjust the force with that the slide strikes the carousel, the spring3046 may be selected to have a different spring constant. In otherwords, the higher the spring constant, the more force the slide willstrike the carousel with. Alternatively, the plunger could be formedwith slots that would allow the screws 3043 to be loosened to adjust theplunger position either towards or away from the carousel to effectivelyadjust the force with which the slide strikes the carousel. The spring3046 is retained in place within the plunger 3024 by the combination ofnipple 3066 and nipple 3064. The diameter of the nipples is to beselected to match the diameter of the spring. As such, the diameter ofthe spring and nipples will likely change to reflect springs havingdifferent spring constants and dimensions.

In an alternative embodiment, the spring 3046 could be substituted witha solenoid that could be electrically activatable to force the slideeither towards or away from the carousel. Such electrical control couldeasily come from the module 3068, in combination, or actingindependently, with computer 3004. Also, if the slide is replaced withthe post as mentioned above, the accuracy of the positioning could beincreased while not increasing the level of human intervention requiredto position the carousel on the housing.

A vial 3010 is shown placed in a port 3008 of the carousel, and alignedwith a receiving coil 3062. Importantly, the slide 3024 holds the vial3010 directly over the antenna coil 3062. While this is not particularlynecessary to insure proper communication between the vial and theantenna coil, the need for accuracy of the positioning increases whenthe distance between the vials is decreased. As discussed above, wherethere is a large number of vials held on a single carousel, there is aneed to properly position the vial so that there is limited, or no,interference between the intended vial and any neighboring vials. Thisproblem would be particularly noticeable in carousals where there isminimal space between the vials, or when the vials have beenminiaturized.

Referring now to FIG. 38, a vial is shown in perspective and generallydesignated 3010. Vial 3010 has three portions: a lid 3076, a cylinder3028, and a sleeve. The vial is shown in cross-section in FIG. 39. Asshown, the lid 3076 is held on the cylinder 3028 by threads 3090 thatare formed on the cylinder and the lid. Although threads provide for aneasy installation and removal of the lid, a snap-type attachment couldalso be used. Moreover, any type of lid could be used and it would notaffect the utility of this system 3000. It will be appreciated by thoseskilled in the art that the ability to add and remove materials from thecylinder is determined by the type of lid that is used. This preferredembodiment uses a lid 3076 that is formed with a membrane window 3078that is preferably made of a puncturable material. This puncturablematerial would allow a syringe to puncture the membrane to inject orremove materials from the cylinder, removing the syringe when finished,allowing the membrane to re-seal itself. Such materials are well knownin the art and are not discussed further here.

The cylinder 3028 is shown filled with material 3050. While this isshown as a fluid, it is to be appreciated that any number of materials,such as polystyrene beads, patient samples and other materials, could beplaced in these cylinders. A sleeve 3048 is attached to the bottom ofthe cylinder 3028 and is formed with an upper orifice 3116 and a lowerchamber 3117. The sleeve 3048 is shown in greater detail in FIG. 40. Theupper orifice 3116 is formed with a circumferential ridge 3110 that isadjacent the upper end of the sleeve. This ridge 3110 is sized to have adistance 3118 between them that is slightly smaller than the diameter ofthe cylinder 3028. This diameter difference is important to insure thatthe sleeve, once positioned, will not slide off of the cylinder. This isparticularly important when it is critical to track the placement andlocation of a vial. In order to achieve the resilience needed to allowthe insertion of the cylinder into the sleeve. The sleeve is fabricatedfrom a suitable insert material, such as polypropelyne material. Whilethis material is fairly pliable, it is also sufficiently rigid to retainits shape. As a result, the polypropylene is particularly suited to suchan application where there is a need to securely mount the sleeve to anobject, while providing sufficient pliability to avoid cracking thecylinder if made of a fragile material such as glass. In situationswhere the cylinder is more rigid and less fragile, the sleeve could beattached using a variety of other manners. More specifically, elasticbands or i-adhesive straps could be used to hold the sleeve in positionover the cylinder.

In order to facilitate the insertion of the cylinder 3028 into theorifice 3116 in the sleeve 3048, a vent hole 3019 is formed in the wallof the sleeve adjacent the divider 3114 of the orifice 3116. This venthole allows the air trapped inside the orifice while the cylinder isbeing inserted to escape into the atmosphere. It is to be appreciatedthat if there were no vent hole 3019 formed in the sleeve, aconsiderable pressure would build within the orifice thereby preventingthe insertion of the cylinder.

On the underside of the sleeve, a chamber is formed between the plug3092, sleeve wall 3112, and divider 3114. Within this chamber is thesleeve antenna 3090, a microchip 3094, both mounted on a substrate 3096.The sleeve antenna 3090 includes multiple windings of a fine gauge,insulated wire to form an inductive proximity antenna. The antenna is animportant feature of the data transmission process between theread/write controller and read/write device. The antenna in thisembodiment has a outer coil diameter of 0.420 inches and an inner coildiameter of 0.260 inches. The coils includes approximately 305 turns ofwire having a diameter of 0.015 inches. In any case, the inductance ofthe antenna once formed should be on the order of 7.92 mH, with a seriesresistance of no more than 850 ohms at 1 volt and 100 KHz. Coil antennasof differing sizes can be used in this embodiment, but the number ofturns of wire will change with the size of the coil, the size of thewire, and the overall diameter of the coil. To determine the propernumber of turns given a different dimensioned antenna, the followingequation must be used:$L = {2*a*{\ln \left( {\frac{a}{D} - K} \right)}*N^{1.9}}$

where “L” is the desired inductance in nH, “a” is the antennacircumference in centimeters, “D” is the wire diameter in centimeters,“N” is the number of windings, “K” is the geometrical constant that fora circular antenna is 1.01, and for a square antenna is 1.47. Becausethe value of “K” for a circular antenna is approximately and is normallymuch smaller than a/D, it can be left out, yielding a simpler equation:$N \approx {1.9\sqrt{\frac{L}{2*a*{\ln \left( {a/D} \right)}}}}$

Thus, once the inductance desired, diameter of the wire andcircumference of the antenna are known, the proper number of windingscan be determined. Or conversely, any one variable can be determinedfrom the equations above to yield the characteristics of the antenna.

In this particular embodiment, the cylinders are vials fabricated fromglass. It is to be appreciated, however, that the cylinder could be madeof virtually any material, including metal or any other inert material.They may also be made of matrices that include at least a portion of asurface suitable for linking biological particles or molecules. In theevent metal is used, there may be a need to elongate the sleeve toinsure that the metal is distanced from the antenna. In addition to thecylinder being made of a variety of materials, it should also beappreciated that any the sleeve can be used with any number ofcontainment devices. Of these devices, the discussed above would be aparticularly well suited container for attachment to the sleeve. Infact, the sleeve could be reformed to a substantially differentstructure that, nonetheless, would work equally as well as the sleeveand cylinder embodiment. Moreover, any other containment device ormatrix support that is provided herein is capable of being equipped withsleeve with memory. It is to be appreciated, however, that particularlysmall-sized read/write device's would require a miniaturized designhaving a smaller sleeve antenna. In fact, in environments where a sleeveis not practical, the read/write device may be made in the form of asubmersible chamber or embedded in the structure of the containmentdevice as described herein.

Also within the chamber 3117 is the microcircuit 3094 and substrate3096. The microcircuit includes a rectifier, voltage regulator, resetgenerator, demodulator, clock extractor, modulator, control unit, andmemory [see, U.S. Pat. No. 5,345,231]. Referring briefly to FIG. 2 inthe U.S. Pat. No. 5,345,231 patent, the overall system architecture ofthe semiconductor is identified. This circuitry derives its power fromrectifying an incoming signal that is received by the antenna. Therectified signal is conditioned with the voltage regulator and fed toall other circuitry on the semiconductor. The microcircuit 3094 isattached to the substrate 3096 using a bonding adhesive as is common inthe art. Any number of attachment methods could be used, however, andwould achieve the same result. The substrate is preferably made ofalumina that would provide a stable platform that would have dielectricconstants approximately equal to those of the silicon wafer of themicrocircuit. As is common in the industry, by matching the dielectricconstants of the materials, as well as the thermal expansioncoefficients of the various materials, there is less of a likelihoodthat there will be cracking on the microcircuit.

Moreover, because the thermal coefficients of the microcircuit areapproximately the same as the alumina substrate, there is a good matchbetween the two that greatly reduces the thermal stresses that arepresent on the microcircuit.

Referring now to FIG. 41, the substrate 3096 is shown with themicrocircuit 3094 firmly attached. In addition to having themicrocircuit mounted to it, the substrate 3096 is also formed with acopper layer that is intended to be the bonding points for the antennaleads 3102. More specifically, the substrate 3096 is formed with a pairof pads 3100. Each of these pads is securely attached to the surface ofthe substrate to allow the wires 3098 from the microcircuit, calledbonding wires, to be easily attached to the antenna wires 3102.Attachment between the two wires is made by soldering or brazing one ofthe wires, preferably the most delicate wire that would be the bondingwire 3102, from the microcircuit to the pad 3100. Once the most delicatewire is attached, the more robust and durable wire is attached to thepad. In this instance, the antenna wire is significantly thicker thanthe bonding wire and, thus, is connected last. In order to simplify theattachment of the antenna wire and to reduce the heat to which themicrocircuit is exposed to, the pad may be tinned. A lead is “tinned”when covered with a slight layer of solder to which the antenna wire canbe more easily attached to.

Once the sleeve antenna 3090 is formed and electrically connected to thesubstrate 3096, the substrate is attached to the antenna using anadhesive 3097. This adhesive may be of any kind known in the art and isselected so that it produces very little radio frequency for otherfrequency when other frequencies are used] interference. In thisembodiment, silicone is used to secure the substrate to the sleeveantenna 3090. It is important to note that the microcircuit 3094 in thepresent embodiment fits nicely within the internal diameter of thesleeve antenna. This is of particular usefulness because suchpositioning effectively reduces the height of the combination of theantenna, substrate, and microcircuit. Once the substrate is firmlyattached to the sleeve antenna, the combination is inserted into thechamber 3117 and held against the divider 3114 by an adhesive. Again,silicone is used to hold the sleeve antenna in place against thedivider. As shown in FIG. 40, there is little space remaining followingthe insertion of the sleeve antenna and substrate into the chamber.

Once the sleeve antenna is positioned within the chamber, the plug 3092is inserted into the sleeve 3048 to seal the chamber 3117. The plug isformed with a circumferential ring 3124 which engages to acircumferential slot 3122 formed in the sleeve wall. The ring and slotengage each other to insure that the plug does not fall out, therebyexposing the delicate electronics to the environment. In an effort tofurther reduce the likelihood of exposure to the environment,particularly when the environment is damaging to the electronics (e.g.wet), the plug can be sealed in place against the sleeve using a heatedtool. Referring to FIG. 42, a heating tool is shown that contains athermally conducting block 3134 that is formed with a hole 3132 forreceiving the sleeve. Specifically, once the plug has been inserted intothe sleeve to seal the chamber, the entire device is inserted into theblock 3134 that has been previously, or currently, heated. Because, asmentioned above, the sleeve is preferably made out of polyethylene, itis easily melted. Thus, by exposing the plug end of the sleeve to a heatsufficient to barely melt it, the plug is fused to the sleeve to createan environment seal. This seal is capable of withstanding even the mostharsh environment, while maintaining the electronics in a dry locationwithin the chamber.

Referring back to FIG. 37, and having review all necessary structuralcomponents of the system 3000, the operation of the system is moreeasily understood. A vial is either filled with a substance, or has beenpurchased with a substance already in it. In either case, this vial isinserted into a sleeve. As the sleeve is inserted over the vial, thepolyethylene yields to provide a solid connection between the sleeve andthe cylinder wall 3028. Preferably, at the time of mating the cylinderwith the sleeve, the microcircuit has no memory stored on it whichrepresents an identification tag. Thus, once the vial and sleeve havebeen combined, the combination is placed in a port 3008 of the carousel3006. Once inserted in the port, the carousel is then placed on thehousing with the notch 3040 adjacent the plunger 3024. Then, thecarousel is rotated such that the vial is directly in front of theplunger, and thus, immediately over the base antenna 3062. Once inposition, the base antenna 3062 is excited with a high frequency (HF)signal from the base antenna 3062 [see, U.S. Pat. No. 5,345,231], whichthereby activates the microcircuit in the chamber 3117. As stated above,this HF signal is rectified and filtered to provide power to themicrocircuit. Additionally, a clock signal is removed from the HF signalradiating from the base antenna, and used to coordinate operationswithin the microcircuit to the operations within the housing and module3068. It should be appreciated that any other manner of exciting anon-powered microcircuit, or accessing identification data from apowered microcircuit, is contemplated by this disclosure. It is alsoappreciated that components suitable for use with other selectedfrequencies are also contemplated herein.

Once rectified, the microcircuit (rwd) is powered on and proceedsthrough its set-up routine which places the microcircuit in a conditionwherein it can be programmed by the read/write controller. Theread/write controller senses that the read/write device is notprogrammed and, as a result, selects an identification number forprogramming. As mentioned above, it is possible to program all of themicrocircuits when the sleeves are made. In such an instance, theprecoded information will be stored in a remote computer and associatedwith information regarding the contents of the vial or container. Thus,when the microcircuit is pre-coded,that identification code inincorporated into a database that identifies the contents of thecylinder for other container] with the identification code. As iscustomary in the industry, a variety of information fields could be usedto accomplish a variety of informational purposes. Specifically, theidentification code programmed into the microchip could be matched witha database that includes all patient information for the samplecontained within the cylinder. For example, if the sample was blood, thedatabase could include the patient's name as well as other aspects ofthe patient's file. In addition, in an automated process, the computersystem where the database is maintained could be continually updated tomonitor the current status of the sample or specimen contained in thecylinder associated with the particular sleeve and identificationnumber.

Once programmed, the sleeve and vial can be removed from the carousel,or the carousel may simply be rotated so that a different sleeve may beinserted into a different port. In this manner, an entire carousel maybe loaded and programmed within a short period of time. Despiterequiring a short program period, the cylinder will never have to bere-programmed. In fact, many of the microcircuits areone-time-programmable (OTP) which removes any problem with erasing theidentification code from the microcircuit. The vials with sleeve may befurther processed in accord with methods described herein or othermethods. The sleeve permits the vials to be readily identified andtracked.

The base antenna is substantially shaped like the sleeve antenna.

Specifically, The ratio between the diameter of the sleeve antenna andthe base antenna should be approximately 1:1. In certain circumstances,the ratio can be as much as 3:1, where there is sufficient distancebetween the vial being identified and the neighboring vials. Matchingthe base antenna to the sleeve antenna is an important aspect of theinductive communication which passes between the two antennas. Moreparticularly, in order for the communication between the two antennas tobe optimized, the antennas must be properly matched and positioned.While it is not critical that the antennas be precisely matched, it isimportant to maintain the ratio of 3:1 or less for the difference insizes between the two antenna.

A pair of leads 3071 pass from the base antenna to a filter board 3072which is intended to adjust the frequency range of the base coil tomatch the frequency range of the sleeve coil. In the present embodimentwhere the base antenna and the sleeve antenna are similarly shaped, thefilter board 3072 only includes a capacitive adjustment. In thisparticular embodiment, the capacitance needed to match the two antennawas on the order of 20 pF.

From the filter board 3072, the electronic signal to and from the baseantenna passes through wire 3080. This wire handles all communicationsbetween the module 3068 and the read/write device. Inside the module isa circuit board which includes those components shown in FIG. 1 U.S.Pat. No. 5,345,231. More specifically, the circuit within the moduleincludes an oscillator, modulator, demodulator, clock extractor, controlunit, and interface.

In particular, the modulator and demodulator are matched to themodulator and demodulator housed on the microchip. The control chipessentially coordinates all activity between the computer system 3004and the module [see, U.S. Pat. No. 5,345,231, which describes theoperation of the circuitry within the module].

Wire 3070 passes from the control module 3068 to the computer system3004. This connection is a standard serial communication channel that isreadily accepted into virtually all modern computer systems. In thepresent embodiment, the computer system is equipped with a program thatis designed to receive the serial digital information from the modulethat represents the identification code of the microcircuit in theread/write device. In addition to decoding the identification number,the computer system can be provided with positioning information thatidentifies the location within the carousel of the particular sleeve andvial. Moreover, because the motor is electrically actuatable, theparticular vial can be identified and repeatedly accessed without havingto re-decode the identification code for each instance.

Although the positioning of the sleeves had been discussed in terms ofbeing mounted on a containment device of one sort or another, it is tobe appreciated that virtually anything could be identified with such aread/write device. More specifically, in the instance of the preferredembodiment, the carousel itself could be equipped with a read/writedevice that would enable the read/write controller to identify whichcarousel out of a number of carrousels was actually installed on thesystem 3000. To this end, it is to be appreciated that the housing 3018could be equipped with a number of base antenna that couldsimultaneously read a number of sleeve antennae and read/write device,as well as those read/write devices located within the carousel itself.

Referring now to FIG. 43, a typical display 3200 is shown that isgenerated from software, such as software that one of skill in the artcould design based on the disclosure herein. As shown, the screen has anumber of graphical plots 3202, 3204 that may be used to show any numberof characteristics of the sample contained within the vial. In thisdisplay, the identification code 3206 is shown at the lower left as readfrom the read/write controller and decoded by the computer system 3004.While this display shows various data fields, it is to be appreciatedthat virtually an unlimited number of data fields 3210 could beincorporated into a series of displays associated or linked with asingle identification code. More particularly, the identification codecould be linked to a database that indicates the contents of thecylinder as well as the location of the cylinder, as well as any numberof other features that are also relevant to the contents of thecylinder. For example, the graphs shown here depict various levels of asubstance which either varies over time, or following a series of tests.

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLE 1 Formation of a Polystyrene Polymer on Glass and Derivatizationof Polystyrene

A glass surface of any conformation [beads for exemplification purposes(1) that contain a selected memory device or that are to be engravedwith a 2-D optical bar code or that include a 3-D memory] that coat thedevice or that can be used in proximity to the device or subsequentlylinked to the device is coated with a layer of polystyrene that isderivatized so that it contains a cleavable linker, such as an acidcleavable linker. To effect such coating a bead, for example, is coatedwith a layer of a solution of styrene, chloromethylated styrene, divinylbenzene, benzoyl peroxide [88/10/1/1/, molar ratio] and heated at 70° C.for 24 h. The result is a cross-linked chloromethylated polystyrene onglass (2). Treatment of (2) with ammonia [2 M in 1,4-dioxane, overnight]produces aminomethylated coated beads (3). The amino group on (3) iscoupled with polyethylene glycol dicarboxymethyl ether (4) [n≈20] understandard conditions [PyBop/DIEA] to yield carboxylic acid derivatizedbeads (5). Coupling of (5) with modified PAL [PAL ispyridylalanine]linker (6) under the same conditions produces a bead thatis coated with polystyrene that has an acid cleavable linker (7).

The resulting coated beads with memories are then used as solid supportfor molecular syntheses or for linkage of any desired substrate.

EXAMPLE 2 Microvessels and Use Thereof

A. FIGS. 11-13

FIGS. 11-13 illustrate an embodiment of a microvessel 20 provided herein[see, also FIGS. 45-50 for an alternative embodiment]. The microvessel20 is a generally elongated body with walls 22 of porous orsemi-permeable non-reactive material which are sealed at both ends withone or more solid-material cap assemblies 42, 44. The microvessel 20retains particulate matrix materials 40 and can, as depicted in theFigure, contain one or more recording devices 34 or alternatively isengraved or imprinted with a symbology, particularly, the 2-D opticalbar code provided herein. In the embodiment illustrated in FIGS. 11-13,the recording device includes a shell 36 that is impervious to theprocessing steps or solutions with which the microvessel may come intocontact, but which permits transmission of electromagnetic signals,including radiofrequency, magnetic or optical signals, to and from thememory engraved on or in the device.

The microvessel 20 is generally cylindrically shaped and has twosolid-material cap assemblies 42, 44. The cap assemblies may be formedof any material that is non-reactive with the solutions with which themicrovessel will come into contact. Such appropriate materials include,for example, plastic, TEFLON, polytetrafluoroethylene (hereinafter,PTFE) or polypropylene. Each cap assembly 42, 44 preferably includes asupport base 26, 28, respectively, and an end cap 24, 30, respectively.Each support base 26, 28 is permanently attached to the walls 22 of thevessel by known means such as bonding with appropriate adhesives or heattreatment, either by heat-shrinking the wall material onto the lowerportions of the support bases 26,28, or by fusing the wall material withthe support base material.

Preferably, at least one of the caps 24,30 is removably attached to itscap base 26, for example by providing complementary threads on thesupport base and the end cap so that the end cap can be screwed into thesupport base, as illustrated in FIG. 12. Other possible means forattaching the end cap to the support base will be apparent to those inthe art, and can include snap rings, spring tabs, and bayonetconnectors, among others. The end cap 24, has one or more slots, bores,or recesses 32 formed in its outer surface to facilitate removal orreplacement, with the user's fingers and/or by use of an appropriatetool. For the example illustrated, a spanner wrench having pegs spacedat the same separation as the recesses 32 can be used by inserting thepegs into the recesses. For a single slot, removal and replacement ofthe end cap could be achieved by using a screwdriver. Protruding tabs,rims, knurled edges or other means to enhance the ability to grasp theend cap can be used for manual assembly/disassembly of the microvessel.The cap assembly 42 at the opposite end of the microvessel can bepermanently sealed using an adhesive or heat treatment to attach thesupport base 28 to the end cap 30, or the cap assembly 42 can be moldedas a single piece, combining the support base 28 and the end cap 30.

Retained within the microvessel 20 are particle matrix materials 40 and,as depicted a memory device 34. In embodiments herein, the memory devicewill be a symbology engraved on the device, such as on the cap.

The illustrated microvessel, as illustrated in FIGS. 11-13, is of a sizesufficient to contain at least one recording device and one matrixparticle, such as a TENTAGEL™ bead. The device is typically 20 mm inlength [i.e., the largest dimension] or smaller, with a diameter ofapproximately 5 mm or less, although other sizes are also contemplated.These sizes are sufficient to contain form about 1 mg up to about 1 g ofmatrix particle, and thus range from about 1 mm up 100 mm in the largestdimension, typically about 5 mm to about 50 mm, preferably 10 mm to 30mm, and most preferably about 15 to 25 mm. The size, of course can besmaller than those specified or larger. The wall material of themicrovessel is PTFE mesh or other chemically inert surrounding poroussupport [polypropylene AA, SPECTRUM, Houston, TX], or other suitablematerial, having a preferably about 50 μM to 100 μM, generally 50 to 70μM hole size that is commercially available. The size of course isselected to be sufficiently small to retain the matrix particles. Thecap apparatus is machined rod PTFE [commercially available from McMasterCarr, as Part #8546K11].

The matrix material is selected based upon the particular use of themicrovessel; for example, a functionalized resin, such as TENTAGEL™resin [e.g., TENTAGEL™ polymer beads carrying an acid-cleavable linker,from matrix material may also include fluophores or scintillants asdescribed herein.

Alternative embodiments of the microvessel will be appreciated andinclude, for example, a pouch, including porous or semi-permeablematerial, which is permanently sealed to itself and contains matrixmaterial and one or more memories.

About 20 mg of the derivatized TENTAGEL™ beads have been sealed in asmall [of a size just sufficient to hold the beads] porous polypropylenemicrovessel (see, Examples, below].

In alternative embodiments, microvessels in which the tube (or othergeometry) is solid (not porous), such as polypropylene, PTFE or otherinert surface that has been radiation grafted, as described herein, mayalso be used. With these devices syntheses are performed on the surfaceand the solid tube is engraved with a symbol or includes an opticalmemory or other tag, which may be permanently or removably sealedinside. These devices, herein denoted the MICROTUBE microreacters (ormicrovessels), may be used in the methods herein interchangeably withthe MICROKAN microreactor type of device. In addition, these may also beadvantageously used in the optical embodiments.

The “microreactors” provided herein permit the advantageous productivitygains of the split-and-pool technique, without any of its limitations.

B. Tagging

By pooling and splitting matrix with memory microreactors [rather thanindividual solid phase resin beads] by a process known as “directedsorting”, one discrete compound is synthesized in each matrix withmemory reactor or microreactor. Each microreactor contains a memory,such as an optical memory, that is a unique label or tag used toidentify it during the sorting processes that occur between chemicalsynthesis steps.

The memory tag provides a unique ID for each matrix with memory reactorand therefore each compound. This unique ID allows each microreactor tobe identified during the combinatorial directed sorting process.

C. The “Directed Sorting™” Approach to Solid Phase CombinatorialChemistry

The “directed sorting” approach to combinatorial chemistry is madepossible by splitting and pooling matrix with memory microreactorsrather than individual solid phase resin beads. During the firstdirected sorting step each microreactor is assigned to one specificcompound. This assignment is maintained during all subsequent directedsorting and synthesis steps.

Tagging with a memory that is either engraved or imprinted duringprocessing, subsequent to or pre-encoded [with decoding informationstored remotely and associated with identifying information] ofmicroreactors provides convenient and positive identification ofcompounds for archival and storage purposes. Such tagging permits themicroreactors to be sorted between the individual steps in thesynthesis.

Traditional split-and-pool methodology relies on a statisticaldistribution of resin beads between each step in the chemical synthesis.Typically, a large number of resin beads are used for each compoundbeing synthesized to ensure an adequate statistical distribution ofcompounds. A consequence of this approach is that individual compoundsare synthesized on multiple solid phase resin beads. These multiplecopies of each compound are mixed together with multiple copies of allthe other compounds. these mixtures need to be deconvoluted duringscreening. In contrast, the directed sorting approach ensures that:

1. Every compound is synthesized

2. Only one copy of each compound is synthesized

3. All compounds are present as discrete entities (no mixtures).

D. Software

Software, described herein, provides:

1. A repository for the chemical synthesis information—primarily thebuilding blocks and reaction steps. Other information, such apre-reaction procedures and reaction work-up procedures ban also bestored.

2. Explicit directions teaching how to sort the microreactors betweeneach reaction step to ensure that all compounds, and no duplicates, aresynthesized.

3. An interface from the chemical synthesis environment and format(individual compounds in microreactors) to the biological screeningenvironment and format (cleaved compounds in 96-well microplates).

D. FIGS. 14-16

FIGS. 14-16 illustrate an alternate embodiment of a microvessel 80provided herein. Like the microvessel described in Example 3, thisembodiment of the microvessel retains particulate matrix materials andcan be imprinted with a symbology or will contain one or more recordingdevices (not illustrated). The microvessel 80 has a single-piece solidmaterial frame 82, including a top ring 84, two support ribs 88, 89disposed diametrically opposite each other and a bottom cap 86. Thesolid material frame 82 may be constructed of any material which isnon-reactive with the solutions with which the microvessel 80 will comeinto contact. Such appropriate materials include, for example, plastic,PTFE, TEFLON or polypropylene, and formation may be by molding ormachining of the selected material, with the former being preferred foreconomy of manufacture.

The sidewall of the microvessel 98 is formed of porous or semi-permeablenon-reactive material, such as PTFE mesh, preferably having a 70 μM poresize. The sidewall is preferably attached to the top ring 84 and bottomcap 86 of the solid material frame 82. Such attachment may be by knownmeans such as bonding with appropriate glues or other chemicals or heat,with heat being preferred.

In the embodiment of FIGS. 14-16, the two support ribs 88, 89 arepositioned opposite one another, however, any number of support ribs,i.e., one or more, may be provided. The microvessel sidewall 98 need notbe fully attached to the support ribs 88, 89, however, the moldingprocess by which the microvessels are formed may result in attachment atall contact points between the frame and the sidewall.

In the preferred manufacturing process, the sidewall material, a flatsheet of mesh, is rolled into a cylinder and placed inside the mold. Theframe material is injected into the mold around the mesh, causing theframe to fuse to the mesh at all contact points, and sealing the edgesof the mesh to close the cylinder.

In the embodiment illustrated in FIGS. 14-15, the microvessel 80 isconfigured with a removable end cap 90. The end cap 90 is preferablyconstructed of the same material as the solid material frame 82. A snapring, or, as illustrated, projections 92, 94 extend downward from theinside surface of the end cap 90. The projections 92, 94 have a flangewhich mates with a groove formed in the inner wall of top ring 84 whenpressed into the top ring to releasable secure the end cap 90 to themicrovessel 80. As will be apparent, other means for releasably securingthe end cap 90 to the top ring 84 can be used, including, but notlimited to, those alternatives stated for the embodiment of FIGS. 11-13.The dimensions vary as described for the microvessel of FIGS. 11-13 andelsewhere herein.

In other embodiments, these vessels fabricated in any desired orconvenient geometry, such as conical shapes. They can be solid at oneend, and only require a single cap or sealable end.

These microvessels are preferably fabricated as follows. The solidportions, such as the solid cap and body, are fabricated from apolypropylene resin, Moplen resin [e.g., V29G PP resin from Montell,Newark Del., a distributor for Himont, Italy]. The mesh portion isfabricated from a polypropylene, polyester, polyethylene orfluorophore-containing mesh [e.g., PROPYLTEX®, FLUORTEX®, and other suchmeshes, including cat. no. 9-70/22 available from TETKO® Inc, BriarcliffManor, N.Y., which prepares woven screening media, polypropylene mesh,ETF mesh, PTFE mesh, polymers from W. L. Gore. The pores are anysuitable size [typically about 50-100 μM, depending upon the size of theparticulate matrix material] that permits contact with the syntheticcomponents in the medium, but retains the particulate matrix particles.

EXAMPLE 3 Manual System

Illustrated in FIG. 17 is a program/read station for writing to andreading from the memory devices in the microvessel. The electroniccomponents are commercially available from the same supplier of thememory devices, e.g., BMDS or ID TAG or the monolithic memory providedherein [Bracknell Berks RG12 3XQ, UK], so that the basic operations andfrequency are compatible. The basic controller 170 and the transceiver172 are disposed within a housing 174 which has a recessed area 176positioned within the transmission range of coil 178. The microvessel180 may be placed anywhere within recessed area 176, in any orientation,for programming and reading functions. Basic controller 170 is connectedto the system controller 182, illustrated here as a functional block,which provides the commands and encoded data for writing to the memorydevice in the microvessel and which receives and decodes data from thememory device during the read function. System controller 182 istypically a PC or lap top computer which has been programmed withcontrol software 184 for the various write and read functions.

An example of the operation of the system of FIG. 17 is illustrated inFIG. 18. When power is supplied to the system, transceiver 172 emits aninterrogation signal 185 to test for the presence of a memory device,i.e., a responder, within its detection range. The interrogation signal185 is essentially a read signal that is continuously transmitted untila response 186 is received. The user manually places a microvessel 180within the recessed area 176 so that the interrogation signal 185provides a response to the controllers indicating the presence on themicrovessel. The system receives the interrogation signal and performs adecode operation 187 to determine the data on the memory device withinthe microvessel, which data may include identification of the device anddata concerning prior operations to which the microvessel has beenexposed. Based upon the data obtained, the system makes a determination188 of whether additional information is to be written. The system thenperforms a write operation 189 to record the immediately precedingoperation. The write operation 189 involves modulating the transmittedsignal as a series of “0's” and “1's”, which are recorded on the memorychip, which typically has a 128 bit capacity. After completion of theprogramming step 189, an error check 190 is performed wherein a secondread signal is emitted to verify the data that was written for integrityand correct content. If the correct data is not verified, the system mayattempt to perform the write operation 189 again. After verification ofthe correct data, if the microvessel is one that should proceed toanother operation, the system controller 182 will display instructions192 for direction of the microvessel to the next process step.

The read operation is the same as the beginning of the write operation,with the interrogation signal being continuously transmitted, ortransmitted at regular intervals, until a response is received. Theresponse signal from the memory device in the microvessel 180 isconducted to system controller 182 for decoding and output of the datathat is stored on the memory device.

Software within the system controller 182 includes a data base mappingfunction which provides an index for identifying the process stepassociated with data written at one or more locations in the memorydevice. The system memory within the system controller 182 will retainthe identification and process steps for each microvessel, and an outputdisplay of the information relating to each microvessel can indicatewhere the microvessel has been, and where it should go in subsequentsteps, if any. After the data stored within the microvessel has beenread, it is removed from the interrogation field and advanced to itsnext process step.

Software for aiding in the steps in combinatorial synthesis schemes hasbeen developed. The software, which in light of the description hereincan be written, facilitates the process of creating chemical librarieswith the systems provided herein. The exemplified software, nowavailable under the name ACCUTAG™ Synthesis Manager Software as a partof the AccuTag™-100 Combinatorial Chemistry System [ e.g., an embodimentof the system provided herein]. These systems exemplified with thedevice of FIG. 17 [e.g., sold under the name ACCUTAG™], computer-basedhardware, and the matrix with memories used therewith, such as theMICROKAN matrix with memory device and the MICROTUBE matrix with memorydevice [see, e.g., FIGS. 11-15 and 21].

The software is organized into the following sections. These sectionspresent the normal sequence of activities that go into building alibrary with the system provided herein.

1. Define Building Blocks. The user enters the names of the chemicalbuilding blocks to be used. For brevity of reference, a code letter isassigned to each building block.

2. Plan Steps.

a. Number of Steps. The user specifies the number of steps. In a givenstep, a building block, such as a monomer, amino acid, nucleotide, willbe chemically added to each compound that is being synthesized.

b. Building Blocks To Use. The user specifies which of the definedbuilding blocks will be used in each step. If, for example, there are 3steps and the user specifies building blocks A, B, C in step 1, buildingblocks D, and number in step 2, and building blocks F, G, H, I in step3, then the resulting library will contain 24 unique compounds becausethere are 3×2×4=24 combinations of building blocks.

c. Procedural information. The user optionally enters “recipe”information such as reaction times, temperatures, molarities, andreagents to use for each building block's reactions as well asprocedures common to all building blocks. At the appropriate timesduring the “Perform Synthesis” section of the program, this informationis “played back” to the user. This is a convenience function for theuser.

3. Perform Synthesis. Using a virtual library database of all theinvolved building blocks, reactions, process and compound tracking data,the software facilitates the step-by-step synthesis of the chemicallibrary using memories with matrices, such as a MICROKAN OR MICROTUBE.For each step specified in Plan Steps (above) the following four tasksare performed.

a. Pre-Procedure. Any preliminary procedures that the user entered aredisplayed. Typically these will involve chemical “deprotection” of thereaction site associated with this step.

b. Sorting. The “directed sorting” process for the current step isadministered by the software. The user is prompted to place a memorywith matrix on the scanning station [see, e.g., FIG. 17], which isconnected to a computer. The memory in the matrix, i.e., the tags,identification [ID] is read. The software does a database look up,seeking this unique ID. On the first step, the tag's ID is not found inthe data base, so the software assigns it to the first compound in thelibrary, which has not yet been associated with a tag. The user isinstructed to place the device into the reaction vessel for theappropriate building block. From this point on, when this tag is read,the user is instructed to put the device into the reaction that will addthe building block planned for this step for this specific compound.

c. Reactions. Through directed sorting, all the devices in the libraryare now in reaction vessels. These is one vessel for each building blockin the current step. The user is now prompted to perform the syntheticchemistry that will add each vessel's building block to the compounds itcontains. The software displays any procedure information pertaining toreaction conditions that the user entered in Plan Steps.

d. Work Up. The user is prompted to perform the “work up” [follow-up]task. Any work-up procedures the user entered in Plan Steps aredisplayed. Typically these involve rinsing and drying the reactordevices.

4. Archive. Archive refers to the process of transferring the completelysynthesized compound from matrices with memories to a storage medium,such as a 96 well microplate or vials of any shape or size.

This works as follows.

a. User chooses either vials or microplates [or other container].

These containers or vials may include memories into which identifyinginformation can be entered, such as by scanning the first memory andthen entering the scanned information into the memory in the matrix[container] into which the compounds are transferred.

b. User places device on memory with matrix reader, a scanning station[see, e.g., FIG. 17].

c. User selects a placement location: a well in a plate or a specificvial number.

d. User affirms placement location and the database is updated todocument this. Chemically, the user typically cleaves the compound fromthe solid phase support and deposits only the synthesized compound inthe storage media, while salvaging the reusable tag device for reuse ona another library.

e. The software automatically selects the next storage location. Theuser may override this, and make another selection.

While not required part of the process, additional functions, such asthe following functions are provided.

1. Utility Functions.

a. Decode Tags. Using this function, at any time, the user can place atag on the Scanning Station. If the tag has been assigned to a compoundin the library, then information about that compound is displayed.

b. Find Compound. The user can specify a combination of building blocks.The software looks up this combination, and if it exists, it displaysinformation about the compound and its tag.

c. Status. Spreadsheets showing all devices, their building blockassignments and process status (which steps have been sorted) is shown.

2. Printing. The user can print out report describing:

a. Building Blocks

b. Steps planned.

c. List of All Compounds.

3. On Line Help. The user can get context-sensitive assistance and ahypertext version of the System's User's Guide.

EXAMPLE 4 Preparation of a Library and Encoding the Matrices WithMemories

A typical matrix with memory, such as the MICROKAN matrix with memoryreactor will provide the following yield:

Resin loading 0.5-1.0 μmol/mg resin Using 30 mg of resin: 15-30 μmolcompound For a 500 MW compound: 7.5-15 mg of compound.

A pool of the matrices with memories prepared as in EXAMPLE 2 was splitinto two equal groups. Each group was then addressed and write-encodedwith a unique radio frequency signal corresponding to the buildingblock, in this instance an amino acid, to be added to that group.

The matrices with memories were then pooled, and common reactions andmanipulations such as washing and drying, were performed. The pool wasthen re-split and each group was encoded with a second set of radiofrequency signals corresponding to the next set of building blocks to beintroduced, and the reactions were performed accordingly. This processwas repeated until the synthesis was completed. The semiconductordevices also recorded temperature and can be modified to record otherreaction conditions and parameters for each synthetic step for storageand future retrieval.

Ninety-six matrices with memories were used to construct a 24-memberpeptide library using a 3×2×2×2 “split and pool” strategy. Thereactions, standard Fmoc peptide syntheses [see, e.g., Barany et al.(1987) Int. J. Peptide Protein Res. 30:705-739] were carried outseparately with each group. All reactions were performed at ambienttemperature; fmoc deprotection steps were run for 0.5 h; coupling stepswere run for 1 h; and cleavage for 2 h. This number was selected toensure the statistical formation of a 24-member library [see, Burgess etal. (1994) J. Med. Chem. 37:2985].

Each matrix with memory in the 96-member pool was decoded using aspecifically designed radio frequency memory retrieving device [BioMedic Data Systems Inc. DAS-5001 CONSOLE™ System, see, also U.S. Pat.No. 5,252,962 and U.S. Pat. No. 5,262,772] the identity of the peptideon each matrix with memory. The structural identity of each peptide wasconfirmed by mass spectrometry and ¹H NMR spectroscopy. The content ofpeptide in each crude sample was determined by HPLC to be higher than90% prior to any purification and could be increased further by standardchromatographic techniques.

EXAMPLE 5 Synthesis of Oligonucleotide Libraries on OMDs

Oligonucleotide libraries are synthesized on OMDS [described above, see,e.g., FIGS. 22-30 and 33]. Referring to FIG. 33, polypropylene sheets[(10×10×1 mm) the Moplen resin e.g., V29G PP resin from Montell, NewarkDel., a distributor for Himont, Italy) are radiation grafted withpolystyrene to give the surface modified devices 1 [MACROCUBES™ orMACROBEADS™]. Each such device is imprinted with a unique symbology,such as the two-dimensional optical bar code using the methods describedherein. The OMDs [also called laser optical synthesis chips] are thensubjected [see, FIG. 33] to a modified aminomethylation procedure[Mitchell et al. (1978) J. Org. Chem. 43:2845; Mitchell et al. (1976) J.Org. Chem. Soc. 98:7357; or other procedures to obtain other functionalgroups(Farrall et al. (1976) J. Org. Chem. 41:3877; Merrifield et al.(1985) Angew. Chem. Int. Ed. Engl. 24:7991)] to functionalize thepolystyrene surface graft. Procedures are exemplified in the examples.

A laser optical memory device, shown in FIG. 33A, was fabricated bycombining two components: a 2-dimensional (2-D) 16 digit bar code forencoding and a separate polymeric support for synthesis. The 2-D barcodes were laser-etched by a CO₂ laser on 6×6 segments of a chemicallyinert alumina ceramic plate (Coors Ceramics, thickness=0.5 mm; theactual size of each 2-D bar code is 3×3 mm). The surrounding synthesissupport is a stable polypropylene or fluoropolymer square (10×10×2 mm)radiolytically grafted (as describe herein) with low cross-linkingpolystyrene (Battaerd, G. W. Tregear, (1967) in Graft Copolymers, JohnWiley & Sons, Interscience, New York) and designed with a square hole(6×6 mm) in the middle. The etched ceramic block is securely insertedwithin the hole to form the entire OMD. Very small size OMDS can bemanufactured as the laser etching optical resolution of an entire 2-Dbar code can extend well below 0.5 mm in total diameter. 2-D bar codinghas the advantage over regular linear bar coding of more datacompression in a much smaller surface area (FIG. 33B).

A loading of a 5-8 μmol/device was typically obtained as measured byFmoc analysis. At this point, the OMDS are ready for use incombinatorial or standard chemical synthesis.

A directed sorting strategy [instead of statistical pool and splitting]was used in the construction of combinatorial libraries with zeroredundancy [i.e., the number of OMDs is equal to the number of thelibrary members]. In an example of a 3×3 directed sorting synthesis [FIG33C), nine OMDs are first scanned optically using a small camera [i.e.,such as the QuickCam™]linked to the pattern recognition software [see,description above and FIG. 31] on a computer, and each device [with aunique 2-D bar-code], i.e., 1-9, for exemplification, is assigned to oneof the nine members in the library [a Code-Structure Table] by thesoftware that directs the synthesis, such as the Synthesis Managersoftware, described herein and in the EXAMPLES]. The OMDs are then split[sorted], using software for synthesis and for decoding the 2-D codepattern [see Appendix I and description herein] into three groupsaccording to the first building block (A, B, or C) for each structure aspre-assigned in the Code-Structure Table. A reaction with building blockA, B, or C is then performed on each specific group. The OMDs are thenpooled, washed, subjected to common reactions, scanned and re-sortedinto three new groups according to the second building block (A, B, orC). A second reaction with building block A, B, or C is then performedwith each group of OMDs. The OMDs can then be pooled again, subjected tocommon manipulations, and sorted. The process is repeated until thesynthesis is completed.

The structure of the compound synthesized on each OMD can then bede-coded simply by optically reading the 2-D image with synthesissoftware via the camera and the decoding software and correlating thebar code with the structures in the Code-Structure Table.

To demonstrate the utility of the laser optical synthesis OMDs in thelarge scale synthesis of oligonucleotides, a library of 27oligonucleotides with a general structure of X₄-X₃-X₂-T was constructedusing the above described Directed Sorting strategy (FIG. 33D). Sincepolystyrene does not completely swell in acetonitrile or water, whichare the solvents for the coupling and cleavage steps in a standardoligonucleotide synthesis cycle[Gait et al. (1990) in OligonucleotideSynthesis, A Practical Approach, Gait, Ed., IRL Press, Oxford], reactionconditions were modified to accommodate the polystyrene support. Amongthe range of solvents and co-solvents investigated, it was found that amixture of acetonitrile and dichloromethane (2:3, v/v) for the couplingreaction gave the highest coupling efficiency, and water/1,4-dioxane(1:1, v/v) performed best as the solvents for the cleavage step. Thestandard conditions for the de-blocking step (3% trichloroaceticacid/dichloromethane) and the oxidation step (0.1 M I₂/THF) are directlyapplicable. All reactions [see FIG. 33D] were performed in appropriatesize glass bottles with Teflon-lined screw caps, and all the washingsare performed using acetonitrile and dichloromethane alternately.

Reaction conditions were as follows: de-blocking, 3% TCA/DCM, rt, 2 min,repeat 2 times, coupling, 0.1 M DMT-X_(n), 0.3 M tetrazole, ACN/DCM (2:3v/v), rt, 1 h; oxidation, 0.1 M I₂, THF/pyridine/H₂O (40:10:1, v/v/v),rt, 20 min; capping, 0.5 M Ac₂O, 0.5 M 1-methylimidazole, 0.45 M 2,6-lutidine, THF, rt, 20 min.; cleavage, concentrated ammonia/1,4-dioxane(1:1 v/v), rt, 20 h; deprotection, concentrated ammonia, 55° C., 20 h,;de-salting (and de-blocking) on Poly Pak cartridges (according tomanufacturer's instructions).

Twenty-seven amino-functionalized OMDs were first reacted with 0.1 M5′-O-DMT-3′-succinic acid-2′-deoxythymidine/0.1 M PyBop/0.2 M DIEA/DMF(30 ml) at room temperature for 4 hours. The OMDs were washed(acetonitrile and dichloromethane alternately, 30 ml×4 for each solvent)and dried under vacuum at room temperature for 30 min (all subsequentreactions were followed by the same washing and drying procedures). TheOMDs were then capped with Ac₂O/1-methylimidazole/2,6-lutidine/THF (0.5M for each, 30 ml) at room temperature for 20 min., and de-blocked with3% trichloracetic acid/dichloromethane (30 ml, rt, 2 min., repeated twotimes). UV measurement of the de-blocking solution indicated an averageloading of 7.0 μmol per OMD. The OMDs were then scanned and each OMD wasassigned to one of the 27 oligonucleotide sequences in the library(Code-Structure Table) using software, such as the Synthesis Managersoftware described herein, and a camera, such as the QUICKCAM camera.The OMDs were sorted into three groups according to the second residueassignment (A, G, or C) and coupled with one of the correspondingβ-cyanoethyl phosphoramidites (0.1 M) in acetonitrile/dichloromethane(2:3, v/v, 10 ml, with 0.3 M tetrazole) at room temperature for 60 min.The OMDs were then pooled together, oxidized (0.1 Ml₂/THF/pyridine/H₂O,30 ml, rt, 20 min), capped, and de-blocked. Next, the OMDs weresubjected to another cycle ofsorting→coupling→oxidation→capping→de-blocking according to the thirdresidue (X₃) in their assigned structures. After the third residue, theOMDs were scanned, sorted into three groups according to their lastresidues (X₄), coupled to the corresponding phosphoramidites, andoxidized. The capping and de-blocking steps were omitted in this cycle.

The washed OMDs were then scanned with the QUICKCAM™ and the sequence ofthe oligonucleotides on each OMD was de-coded. Each OMD was put into a 5ml glass vial labeled with the corresponding oligonucleotide sequence.Concentrated ammonia and 1,4-dioxane (2 ml, 1:1, v/v) was added to eachvial and the vials were sealed with Teflon-lined screw caps. The vialswere shaken at room temperature for 20 hours to cleave theoligonucleotides from the support. The OMDs were then removed from thevials and rinsed with aqueous dioxane (0.5 ml×2) and the vials wereevaporated to dryness under vacuum. Next, fresh concentrated ammoni a (2ml each) was added to each vial. The vials were capped tightly andheated in an oil bath (55° C.) for 20 hours to removed the cyanoethyl,isobutyryl, and benzoyl groups. An aliquot (10 μl) of the crudeoligonucleotide solution from each vial was saved for HPLC analysis andthe rest of the solutions were evaporated under vacuum. The crudeoligonucleotides were then de-blocked and de-salted with Poly Pak™cartridges (Glenn Research) using standardized procedures[User Guide forPoly Pak Cartridges from Glenn Research]. The fully de-protectedoligonucotides were lyophilized from 20% acetonitrile/water (whitesolids), weighed, and analyzed by MS, ¹H NMR:

[Selective ¹H NMR spectra data (500 MHz, D₂O). ACCT: s=8.47 (s, 1 H,CH-adenine), 8.37 (s, 1 H CH-adenine), 8.09 (d, J=7.8 Hz, 1 H,CH-cytosine), 8.06 (d, J=7.8 Hz, 1 H, CH-cytosine), 7.68 (s, 1 H,CH-thymine), 6.15-6.32 (3 multiplets, 6 H, CH-cytosine and O—C(N)H),3.81-5.03 (6 multiplets, 16 H, O—CH₂ and O—CH), 2.28-2.92 (4 multiplets,8 H, CH₂), 1.87 (s, 3 H, CH₃). ACAT: s=8.47 (s, 1 H, CH-adenine), 8.44(s, 1 H CH-adenine), 8.35 (s, 1 H, CH-adenine), 8.34 (s, 1 H,CH-adenine), 8.05 (d, J=8.0 Hz, 1 H, CH-cytosine), 7.59 (s, 1 H,CH-thymine), 6.15-6.43 (3 multiplets, 5 H CH-cytosine and O—C(N)H),3.78-5.1 (7 multiplets, 15 H, O—CH₂ and O—CH), 2.17-2.87 (5 multiplets,8 H CH₂), 1.79 (s, 3 H, CH₃).]

The OMDs were also analyzed by sequence analysis. Oligonucleotidesequence analysis was performed using electrospray and EM massspectrometry [ES MS-MS; see, e.g., Siuzdak (1966) in Mass Spectrometryfor Biotechnology, Academic Press, San Diego; Metzger et al. (1994)Anal. Biochem. 219:261; Ni et al. (1996) Anal. Chem. 68:19891.

Each oligonucleotide gave the expected molecular ion in massspectroscopy analysis as configured by the 2-D encoding. Sequenceanalysis of two oligonucleotides with the same molecular weight showedthat they had the expected sequences. The crude products had good toexcellent purity as analyzed by reverse phase HPLC. A quick, standardde-salting procedure using Ply Pak cartridges yielded 27 pure (>95% byHPLC) oligonucleotides with good overall isolated yields (while solid,2-7 mg each). These data show that the combinatorial strategy ofoligonucleotide synthesis using the laser optical synthesis (LOSC)technology should produce a large number and quantity ofoligonucleotides with high purity.

EXAMPLE 6 Radiation Grafting of a Polymer on a Insert Surface forPreparation of Matrices With Memories

Matrices for use as supports for synthesis and for use in coupled[single platform] protocols have been prepared using radiation grafting.These supports include any inert surface, including PFTE [TEFLON™],which heretofore does not appear to have been used for radiationgrafting. The methods exemplified below with reference to FIG. 34 havebeen designed for use with PFTE as well as other surfaces. A method ofradiation-induced grafted copolymerization of styrene to Teflon (PTFF)has been developed.

A. Scheme 1

1. Preparation of polymer

Scheme 1 shows the preparation of polymer. Polystyrene is radiationgrafted onto polypropylene or TEFLON® tubes, an RF tag, such as the BMDStag, or IDTAG transponder, was inserted into the tube to produce whatwill be provided under the name MICROTUBE.

The polystyrene is then functionized with selected functional groups[i.e., such as “A” inf FIG. 34A]. Scintillant is covalently linked ontothe polystyrene though “A”, and a bioactive molecules, such as, forexample, biotin, can be synthesized on the surface using the remaining“A” functionalities.

2. Radiation

The teflon (PTFE) tube was radiated under a Co⁶⁰ source at a dose rateof 0.1×10⁵ r/h; the total dose is typically 2.6-2.9×10⁶ r.

3. Polymers

Using radiation-induced grafting polymerization techniques, a variety ofmonomers such as styrene, acrylic acid, methylacrylic acid,2-hydroxymethylacrylate, and other such monomers can be used to producedifferent polymeric surfaces with different functional groups onpolypropylene (PP), polyethylene (PE) and fluoropolymers. Polyethyleneoxide (PEG) may be grafted onto the surface to change the hydrophilicityand reduce the sterichinderance to antibodies or receptors. Functionalgroups such as amines, alcohols and phenols, carboxylic acids, halides,aldehydes, nitrites and other such groups, can be introduced.

It was found that dilution of monomers, such as styrene, with methanolenhanced the rate of grafting PP and PTFE tubes have demonstratedhighest styrene grafting at styrene concentrations of about 25 to 50%.

4. Functionalization

The functionalization was performed using the readily availableN-(hydroxymethyl) phthalimide, with trifluoromethanesulfonic acid ascatalyst. The polystyrene grafted tubes is thoroughly washed before useto remove residual monomer, non-attached polystyrene and additivesremaining from radiation grafting. The amidoalkylation proceeds smoothlyin the 50% (v/v) trifluoroacetic acid—dichloromethane as solvent at roomtemperature for 24 hours. The predetermined loading can be obtained bychanging the concentrations of reagent, catalyst and reaction time. Thehydrazinolysis in refluxing ethanol gives the aminomethyl polystyrenegrafted PTFE tube. The microtubes were prepared in different sizes (2-12mm) with loading capacity range from 0.5-15 μmol per tube.

5. Fluorophores

The scintillants, which are chemical stable, were chosen to match theenergy gap from radiation energy of radioisotopes. Scintillants such as9-anthracenepropionoc acid, 1-pyrenebutanoic acid and their derivativesare matched to the energy transfer for different radioisotopes, inincluding ¹²⁵I, ³H, ¹⁴C and others. Care should be taken when selectingcombinations of scintillants and radioisotopes to match so that energytransfer from isotope to scintillant is matched.

A portion of the functional groups were covalently linked to the mixtureof primary fluor (S1, molecules that emit light following interactionwith radiation) and secondary fluor (S2, wavelength shifter).Experiments were performed with mixture of S1/S2 at the ratio rangingfrom 20:1 to 100:1 for S1 and S2 respectively, with optimum ratio of40:1 for most of the experiments presented here. Conditions in which 20%to 80% of the functional groups were occupied with mixture of S1/S2 wereevaluated. The optimum number of the functional group linked to primaryand secondary fluors for most of the experiments was 50%.

The remaining of the functional groups (20% to 80%) were used forchemical synthesis. Small molecules (e.g., biotin) were synthesized onthe solid support as described in the scheme 2.

B. Scheme 2: Biotin synthesis

In order to reduce steric hinderance and improve the interaction oflabeled biological target (e.g., ¹²⁵I-receptor), and depending on thesize and nature of the small molecule, a different percentage of thefunctional groups was utilized for chemical synthesis, while theremaining functional group were blocked with Boc. The experimentsindicate that optimum results are obtainable with 25% of the functionalgroup dedicated for chemical synthesis.

1. Synthesis

Fmoc (Fmoc-Gly-OH) and Boc(Boc-Gly-OH) linked amino acids were used tocontrol the loading of scintillants and remaining amines. The Fmocgroups were removed using 20 piperidine in DMF, and Boc groups wereremoved using 1:1 ratio of TFA and dimethylmethane. 50% amine groupswere covalently linked to scintillants. The remaining 50% amine wereused to synthesis biotin.

2. SPA Assay

The biological activity of small molecules synthesized on the surface ofthe tubular matrices with memories may be evaluated in a variety ofscintillation proximity assay formats as described herein. For example,biotin and its derivative (2-imidazolidone-4-carboxylic acid) weresynthesized on the tube and the binding characteristics of thesynthesized molecules on the solid support to 125I-streptavidin inscintillation proximity assay were evaluated. The results demonstratedthat biotin derivative (2-imidazolidone-4-carboxylic acid) that has muchlower affinity for streptavidin exhibited a lower signal.

C. A a teflon tube [19 mm, long, OD:5 mm, ID:2 mm; see FIGS. 34C and34D] is radiation grafted. It was found that dilution of styrene withmethanol enhances the rate of grafting. Dilutions of from 5% to 70% weretested. The PTFE tube has the highest styrene grafting at a 50% dilution[in contrast, a polypropylene tube has the best performance at 35%dilution. The teflon (PTFE) tube is radiated under Co⁶⁰ source at a doserate of 0.1×10⁶ r/h; the total dose of 2.6-2.9×10⁶ r.

Functionization is performed using N-(hydroxymethyl) phthalimide, withtrifluoromethanesulfonic acid [TFMSA] as a catalyst. The polystyrenegrafted PTFE tube is thoroughly washed before use to remove residualmonomer, non-attached polystyrene and additives remaining from radiationgrafting. The amidoalkylation proceeds smoothly in the 50% (v/v)trifluoroacetic acid-dichloromethane solvent at room temperature for 24hours. The predetermined loading can be obtained by changing theconcentrations of reagent, catalyst and reaction time. Thehydrazinolysis in refluxing ethanol gives the aminomethyl polystyrenegrafted PTFE tube.

FIG. 34 depicts the protocol for radiation grafting of polymers to thesurface of TEFLON® [or other suitable surface]. FIG. 34C depicts thepreparation of a tubular devices in which the matrix is the radiationgrafted PTFE and the memory is a transponder, such as the monolithicdevice provided herein, the BMDS transponder or IDTAG™ transponder [suchas a MICROTUBE], described herein; and FIG. 34D depicts the small chip[2 mm×2 mm×0.1 mm, see, FIGS. 51 and 51] encased in a radiation graftedpolyprolene or teflon ball [or bead, conical tube or other suchgeometry] with a screw cap or snap on lid [such as device providedherein that will be provided under the name MICROBALL or MICROBEAD orMICROTUBE]. These devices may have removable lids, such as a snap onlid, preferably a snap on lid, or a screw top, so that the memory devicecan be removed and reused, and can be added after radiation grafting.

Loading on the grafted tubes and balls is adjustable can was typicallyabout 0.5-15 μmol per tube. The amount can be varied by altering thesize of the tube or balls. A variety of selected functional groups areavailable. Any known to those of skill in the art may be used, includingany described herein.

PFTE devices are particularly suitable for high temperature reactions[loading was less than or about 3 μmol per device].

EXAMPLE 7

An exemplary glucose sensor with memory is set forth in FIGS. 54-59 anddescribed in this example. This sensor unit which includes a memory maybe programmed to collect data on glucose levels at a user definedschedule and to transmit the collected data to an external reader ondemand, providing instant access to the most recent glucose history forthe patient. In addition, the patient's prescribed treatments (e.g.,insulin injections) can be stored in the sensor along with informationabout actual insulin injections and may be retrieved remotely. Thisinformation is available to the attending physician even without accessto the patient's usual records. This can be very useful, for example, incases for very young or old patients or for patients who are impaireddue to hypoglycemia.

The most common method of measuring glucose is to use the enzyme glucoseoxidase (GOx) to catalyze the reaction of glucose and oxygen to formhydrogen peroxide and gluconic acid:

glucose+O₂→^(GOx)→H₂O₂+ gluconic acid

Given a sufficient oxygen supply, the concentration of glucose isproportional to either the consumption of oxygen or the production ofhydrogen peroxide.

Hydrogen peroxide, however, tends to inactivate the glucose oxidasethereby shortening the sensor lifetime. To remedy this, the enzymecatalase can be used to break down the hydrogen peroxide

H₂O₂→^(catalase)→½O₂+H₂O

so that the overall reaction is

glucose+½O₂→^(enzymes)→gluconic acid

Because of the very strong buffering capacity of physiologicalsolutions, any pH change from this reaction is typically very small.

Incorporation of this process for use as a sensor for detection ofglucose concentration requires two oxygen sensors. The first oxygensensor measures the ambient oxygen concentration surrounding the sensor.The second oxygen sensor is covered with a membrane or gel layer thatcontains the enzymes glucose oxidase and catalase. Oxygen and glucosediffuse into this layer and react according to the above reactions. Anyexcess oxygen remaining after the reaction is detected by the secondoxygen sensor. The glucose concentration is proportional to the decreasein oxygen as measured by the difference between the two sensors.

A drawback of this method is that the oxygen concentration in livingtissue is roughly two orders of magnitude less than that for glucose(0.01-0.15 mM for oxygen to 1-20 mM for glucose). Thus, once theavailable oxygen has been consumed, the above reaction is no longersensitive to changes in glucose. This has been termed the “oxygendeficit” problem.

To address the oxygen deficit, sensing methods have been devised that donot rely on the use of glucose oxidase. Such methods include: opticalmethods (IR absorbency, competitive dextran binding) or the directelectrocatalytic consumption of glucose. Attempts have been made to“replace” the function of oxygen by using either electron carriermediators, such as ferrocenes, or by directly coupling the glucoseoxidase to the electrode in “wired” electrodes.

There are numerous techniques for dealing with the oxygen deficit bylimiting the diffusion of glucose relative to that of oxygen. Forexample, in one method a special membrane is placed over the enzymemembrane that limits glucose diffusion. A second technique uses ageometrical arrangement that allows oxygen to diffuse everywhere butonly allows glucose to diffuse through a small opening.

The process of sensing oxygen in a solution is commenced by a chemicalreaction initiated electrically by one or more electrodes. When thepotential of a noble metal electrode, e.g., Pt and Au, is made suitablynegative, e.g., −500 mV with respect to a solution, oxygen at thesurface of the electrode is reduced by the following reaction:

O₂+4H⁺+4e⁻4e⁻→2H₂O.

The removal of oxygen leads to the diffusion of oxygen to the surface ofthe electrode. The electrode current is proportional to the flux ofoxygen with

Flux=D(Δ[oxy]/ΔL)

where Δ[oxy] is the difference in oxygen concentration between theelectrode and the surrounding media or bulk concentration. Because theconcentration of oxygen at the electrode is zero, [oxy] is equal to theoutside bulk oxygen concentration. ΔL is the distance from the electrodesurface at which oxygen is depleted to where the oxygen concentration isequal to the bulk oxygen concentration (the oxygen gradient boundarylayer region) and D is the constant of proportionality or diffusionconstant for oxygen in an aqueous media. If the boundary layer, ΔL, isheld constant then the electrode current is proportional to the oxygenconcentration alone.

To maintain a constant potential between the solution and the oxygensensing electrode, a three electrode potentiostat is used which containsa working electrode (where the oxygen is reduced), a reference electrode(which maintains a constant potential with respect to the solution), anda counter electrode. Because current flow through the referenceelectrode compromises its function, a counter electrode is provided as acurrent return path for the current flowing through the workingelectrode.

Glucose Sensor #1

FIG. 54 provides a diagram of a simplified circuit, with workingelectrode 542, counter electrode 544, reference electrode 546, andamplifiers 548 and 549. Working electrode 542 is typically platinumbecause it provides the most stable long term operation. Gold is also acommon choice. Reference electrode 546 is usually made of silver with acoating of silver chloride to form a conventional silver/silver chlorideelectrode. For convenience, the counter electrode 544 is usually made ofthe same material as the working electrode, e.g., platinum or gold.

The oxygen reduction and the glucose oxidase reaction are temperaturedependent. Therefore, for accurate measurements, it is necessary tomonitor the temperature and compensate for any changes in temperature.To measure temperature, a thermistor chip is placed in close proximityto the sensing electrodes. The thermistor circuit 550 is a simpleconstant current circuit with changes in the measured voltageproportional to the changes in resistance of resistor 552 caused bytemperature, as illustrated in FIG. 55.

FIG. 56 is a block diagram of the major components of an exemplaryimplantable glucose sensing system, which may be broken down broadlyinto the electrical components 560 and the sensor electrodes 576. Thevarious components are discussed in more detail below. FIG. 57 is across-section of an exemplary implantable device with electroniccomponents on the top half of the printed circuit board (PCB) 572 andthe batteries 574 on the bottom with the sensor electrode assemblyprotruding 576 from one end of the PCB 572.

The PCB 572 may be on the order of about 50 mm×50 mm (2″×2″), but ispreferably smaller.

The basic electronic circuit is provided in FIG. 54. A perspectivediagrammatic view of the electrode section 576 of the electrode sectionof the sensor assembly 576 is provided in FIG. 58. The electrodes542,544,546 are formed by photolithographic patterning of thin-filmmetals deposited on both sides of a silicon or ceramic substrate 588using conventional semiconductor processing techniques such asevaporation or sputtering. The substrate 588 is thin 10.2 mm (0.01″) orless], also in accordance with standard semiconductor processingtechniques. Where silicon is used, the substrate 588 is preferablycoated with a silicon nitride (Si₃N₄) layer for passivation. Theapproximate dimensions of the base metal electrodes, on both sides ofthe substrate 588, in the exemplary embodiment are as follows: counterelectrode 544: thickness of 100 Å Ti/3000 Å Pt, 0.13 mm (0.005″) wide by1.78 mm (0.07″) long; working electrode 542: thickness of 100 Å Ti/3000Å Pt, 0.08 mm (0.003″) wide by 1.78 mm (0.7″) long; reference electrode546: thickness of 100 Å Ti/5000 Å Ag/2000 Å AgCl, 0.13 mm (0.005″) wideby 1.78 mm (0.07″) long, space apart from each other by 1.0 mm (0.004″).The substrate 588 dimensions are approximately 2 mm long (0.078″), 0.8mm (0.03″) wide, and 0.2 mm (0.008″) thick, including the passivationlayers. In each electrode, 100 Å titanium layer is provided foradhesion. In the reference electrode 586, the top 3000 Å of silver isconverted to silver chloride by exposing the silver layer to a diluteferric chloride solution.

The electrodes 542,544,546 are connected to the main circuitry by meansof tape automated bonding (TAB) a polyimide flexible circuit 5810 to thesensor and the main PCB 5812. (Note that the actual conductors are notvisible in the figure since they are sandwiched between the substrate582 and the flexible circuit 5810. A low viscosity polymer encapsulant5812 is used to encapsulate the connection between the flexible circuit5810 and the metal electrodes 542,544,546. A hydrophilic electrolytemembrane 5814 is deposited onto the metal electrode surfaces on bothsides of the substrate 588. Electrolyte membrane 5814 contains aphosphate buffer, pH 7.4 solution to provide stable contact between thecounter, working, and reference electrodes 542,544,546. Electrolytemembrane 5814 is typically polyhydroxyethylmethacrylate orpolyacrylamide. The membrane 5814 is soaked in the phosphate buffer.

The entire assembly, excluding the tape connections to the PCB 572, isencased in a sheath 5816 of silicone rubber. A cavity 5818 over themetal electrodes 542,544,546 is formed on the top side of the substrate588. Finally, the cavity 5818 is filled with a glucose oxidase/catalasemembrane 5820. Membrane 5820 is composed of an aqueous solution ofalbumin, glucose oxidase and catalase which is crosslinked in a 25%glutaraldehyde solution.

Given the close proximity of electrode assembly 542,544,546 and PCB 572,it is sufficient to measure the temperature on the PCB. This isaccomplished by use of a standard surface mount NTC thermistor 552 suchas a NHQ 103R10 from Thermometrics, which is run using the constantcurrent circuit illustrated in FIG. 55.

Referring back to FIG. 56, a microprocessor 562 which is used to controlthe sensor must be selected for low power operation and includemulti-channel analog-to-digital capability. The PIC 16C71 is a low powermicroprocessor running at 32 kHz with four 8-bit AID channels. Separate,interconnected integrated circuits can be used for the microprocessorand the A/D converter, however these will likely occupy a larger area ofthe PCB, limiting the miniaturization of the sensor.

When activated, the microprocessor 562 first checks for requests fordata from the external transmitter (not shown) that are being receivedvia antenna 564 and RF modem 566. If no request is present,microprocessor 562 reads the data from the three input channels, i.e.,the two oxygen sensors and the thermistor 542,544,546, computes aglucose concentration and stores these values in memory 568. The memory568 operates in a scrolling format such that the oldest written data islost. The software flow is provided in FIG. 59.

To conserve power, microprocessor 562 is turned off when inactive and nodata storage or data reading is being conducted. A special, very lowpower (0.1 A) capacitive charging circuit 565 is used to time sampling.It is understood that one of skill in the art could readily, in light ofthe disclosure herein, substitute a software-based timing controller forthe capacitive charging circuit. When the circuit 565 is sufficientlycharged, microprocessor 562 is activated. Circuit 565 is preferablytuned to a frequency of between 10-20 seconds per sample.

As illustrated in FIG. 59, in step 592, the sensor's power is turned onin response to a signal initiated by the sampling timer 565. If a signalis received from the external reader (step 594), microprocessor 562 willfirst provide a signal to the RF modem 566 and antenna 564 to transmitthe sensor's identification and basic status, such as serial number,offsets and calibration data [e.g., temperature corrections (step 595)].Then, in step 596, the contents of memory 568 are transmitted, afterwhich the microprocessor 562 again checks to see if additional data isrequested by the external reader (returning to step 592). If no externaldata request is made, the current readings from the electrodes arecollected and converted by microprocessor 562 into digital data in step598. (The signal generated by the electrodes is analog.) In step 5910,the digital data is stored in memory 568. If all memory locations arefull, the oldest data will be overwritten. After writing, the sensor'spower is turned off until the next timing cycle begins.

Collected data will be stored in memory unit 568, which can be either a2 KB EEPROM unit, such as a Microchip 24C16, or several 256 byte RAMunits. The EEPROM uses less space but more power than the RAM chips. TheEEPROM retains its memory even without power and, thus, does not createa power drain when microprocessor 562 is powered down. (Memory retentionfollowing complete loss of power, i.e., a dead battery, is of littlevalue since an implant without power is no longer useful.) As previouslydescribed, a wireless transmitter is used to transfer data collected bythe sensor to a remote reader. In FIG. 56, transmitter 5610 isillustrated as an antenna coil 564 and RF modem 566 combination, but maybe any antenna configuration designed to transmit the contents of memoryalong with implant specific data such as an identification number,offsets and the like, in response to an inquiry signal from an externaltransceiver (not shown) or at regular time intervals. Transmitter 5610has a range of at least 152 mm (6″) between the implant and externaltransceiver.

Power for all sensor functions is supplied by one or more encapsulatedbatteries 574, shown in FIG. 57, which may, for example, be the lithiumthin package 3CXM 3V battery from Gould Electronics. Preferably, amagnetic switch 5616, shown in FIG. 56, is included to allow the battery574 to be disconnected from the rest of the circuit during inactive orstorage periods, as determined by the sleep timer 565. The battery 574and the magnetic switch 5616 may be mounted on the underside of PCB 572.(Only the battery 574 is illustrated in FIG. 57.)

The electronics portion of the implant is encapsulated within an inert,non-porous material 578 to protect it from the external environment. Inthe exemplary embodiment, the implant is placed in a mold fixture withthe outward end of the sensor assembly with the sensor electrodes 576protruding from the mold. The mold is filled with a low viscosity epoxysuch as Stycast 1266 from Emmerson and Cummings. A vacuum is pulled onthe mold to remove any entrapped air bubbles.

In order to minimize adverse tissue response to the implant, the implantis coated with a biocompatible matrix material 5722 that is known to begenerally inert and well tolerated by the tissues. Examples of thesematerials are silicone rubber or a collagen/albumin membrane. Asdescribed elsewhere herein, an angiogenic factor, such as a growthfactor or interleukin, may also be incorporated into the matrixmaterial.

Fabrication of the exemplary sensors includes a first stage of mountingthe various integrated circuits and other discrete components, showncollectively in FIG. 57 as surface mount components 560, which includeall of the electronic components of 560, except for the battery 574. Thebattery 574 is attached to the underside of the PCB 572, but is not yetconnected. Correct operation of the surface mount components 5710 istested using an external battery supply (not shown) to preventunnecessary battery drain.

The second stage of fabricating the sensors is formation of the sensorassembly [electrode] 542,544,546, as previously described, up to theassembly step prior to addition of the glucose oxidase/catalasemembrane. The electrodes 576 are then attached to the PCB 572 usingpolyimide tape 5712 in accordance with conventional TAB techniques. Thisattachment means provides a good electrical connection while providing alevel of flexibility between the electrodes 542,544,548 and the rest ofthe implant. Again using an external power source, a “burn in” processis run on the sensors to stabilize them and test for proper performance.

After correct operation of all components is verified, the battery 574is connected and the assembly is tested again to check for proper systemperformance. After testing, the assembly including the PCB 572, surfacemount components 560 and battery 574 is encapsulated as described asabove, with the electrodes 542,544,546 left outside of the mold.

The glucose oxidase/catalase membrane 5820 is added into the cavity 5818in the silicone rubber sheath 5816 over the top electrodes using aglutaraldehyde crosslinking solution. After the membrane 5820 iscrosslinked, the complete implant is rinsed in sterile solution. Thematrix material 5822 is then added by spray or dip coating the entireouter surface of the implant except for the axial end of cavity 5818where the glucose oxidase membrane 5820 contacts the solution. Aftercompletion, the unit is sterilized using gamma or e-beam radiation orETO. From this point, the implant must be handled under sterileconditions. A pre-implant calibration is performed and this informationis downloaded into the implant memory, making the implant ready forimplantation.

EXAMPLE 8 Intracranial Pressure Monitor

Monitoring of intracranial pressure is indicated when intracranialswelling presents the risk of injury or death in acute situations (headtrauma) and chronic conditions (hydrocephalus or brain tumor). Theprimary methods of intracranial pressure monitoring, includingintraventricular catheter, subarachnoid screw or bolt, and epiduralsensor, are invasive. The monitor is inserted through the skull, andrequires an external, wired connection to a storage and display device.While most acute situations require only short-term monitoring wherehard-wired connections are generally tolerated, chronic conditions,which would be better controlled with regular, long-term monitoring,present more of a problem.

The pressure sensor provided herein solves this problem. In a mannersimilar to the glucose monitoring embodiment of the recording device, apressure sensor that produces an electrical signal is connected to anelectrical circuit similar to that illustrated in FIG. 56, i.e.,electrical assembly 560, with the distinction being that the electrodesare replaced by a pressure microtransducer 602, as shown in FIG. 60.Such microtransducers for Iintracranial pressure monitoring are knownand are commercially available from a number of different manufacturers:Camino Laboratories of San Diego, Calif., Codman & Shurtlef Inc. ofRandolph, Mass., and InnerSpace Medical of Irvine, Calif.

This microtransducer is typically an optical pressure sensor thatincludes a small displacement diaphragm 604 in front of two opticalfibers. Light is supplied through one of the fibers and is reflected bythe displacement diaphragm into the second fiber where it is directed toan intensity-sensitive optical detector near the end of the secondfiber. The intensity of the reflected light provides an indication ofthe amount of displacement of the diaphragm. Since thecommercially-available optical transducers are still typically disposedwithin the end of a catheter that is connected to some external device,in order to make the sensor independent of external physicalconnections, an LED flight emitting diode) light source 606, and amulti-wavelength optical detector 608, are included on the PCB 6010 withthe other drive electronics, logic and communication (telemetry)components, so that the LED 606 and the optical detector 608 are inoptical communication with the optical fibers 6012 and 6014, and so thatno external connection is required.

Other non-optical pressure transducers are available and can besubstituted for the above-described optically-based pressure transducer.For example, the NPC-109 disposable medical pressure sensor from LucasNova-Sensor would be a suitable alternative. In such embodiment, thesensing element changes ersistance with pressure and one member of awheatstone bridge. The measurement is made by applying a fixed voltagee.g., 4 volts, and measuring the output voltage, which will be on theorder of about 30 microvolts per millimeter mercury.

The microtransducer is mounted inside an axial bore through a small bonescrew 614, as illustrated in FIG. 61, so that the transducer is exposedthrough a small channel 616 in the tip of the screw 614. The back end618 (not shown) of the screw holds the necessary drive electronics,logic and communication (telemetry, i.e. the RF or other remotecommunication circuitry) components, including, the LED light source 606and optical detector 608.

The intracranial pressure sensor, contained within screw 614, isinserted through a burr hole that is drilled through the patient's skullso that the channel 616 at the tip is exposed to the desired locationwithin the skull, depending on the desired monitoring site (ventricular,subarachnoid, or epidural).

As with the glucose monitor, the frequency of data collection can beuser-defined by pre-programming the microprocessor, with the data beingstored in the sensor's memory until the data is requested by thephysician using an appropriate remote transceiver.

Other types of pressure monitors may be used in place of themicrotransducers, such as strain sensors. Optical strain sensors useseveral methods including optical fiber Bragg gratings (FBG), stimulatedBrillouin scattering, and polarimetry in birefringent materials. The FBGis the most readily available type of strain sensor, and is formed bywriting a Bragg-type refractive index grating 624 into a single-modegermanium-doped silica fiber 622, as shown in FIG. 62. (The Bragggrating is shown diagrammatically as a sawtooth pattern.) Strain isdetected by monitoring the reflected wavelength from the grating 624 asit is subjected to elongation or compression. As above, for a remotelyaccessible sensor, an LED (light emitting diode) light source 626, and amulti-wavelength optical detector 628, are included on the PCB 6210 withthe other drive electronics, logic and communication (telemetry)components 6212, so that the LED 626 and the optical detector 628 are inoptical communication with the grating 624. Since FBGs are temperaturesensitive and compensation may be required, the same temperaturemeasurement circuitry as provided for the glucose sensor embodiment isincluded.

Optical pressure sensors based upon microbending are also known and canbe constructed with an optical fiber sandwiched between two opposingserrated plates that bend the fiber when pressure is increased, causingan intensity loss in light transmitted through the fiber. Again, such asensor requires a light source and a light detector in order to be ableto function without external physical connection. Fabry-Perotinterferometric devices are also known for their ability to measurepressure changes. These can be constructed by bonding a silicon membraneonto the end of an optical fiber, and are available from Photonetics,Inc. This particular type of sensor requires two light sources withdifferent wavelengths which, although making the device quite accurate,may impose a limitation on the ability to miniaturize the device.

EXAMPLE 9 Blood Urea Sensor

Chronic dialysis treatments for patients with kidney disease must bemonitored to determine their efficacy in removal of toxins, includingurea, from the patient's blood. Current monitoring methods require thepatient's blood to be drawn for laboratory processing to measure ureaconcentration, with the data being processed either manually or bycomputer before any adjustments can be made in the treatment.

Although there are about thirteen dialysis treatments each month, bloodchemistries are typically measured only once a month, resulting in longdelays in modifying therapy prescriptions for patents. Also, eachtreatment session cannot reveal changes in the patient's condition thatoccur throughout the month. A device for real time monitoring of bloodurea level during dialysis would permit individualized prescription andnutritional therapy for patients on hemodialysis and reduces the needfor additional blood samples and laboratory analyses.

To provide such a device, the logic, power and communications (i.e.,telemetry) components, as previously described, are combined with a ureasensor which is connected in-line with the dialysate effluent comingfrom the hemodialysis cartridge. The decrease of urea throughout thetreatment can be correlated to the amount of urea removed.

As shown in FIG. 63, the urea sensor includes a pair of pH sensors632,634 mounted in a “T” connector 636 that is placed between thehemodialysis cartridge dialysate output port 638 and the normal tubing6310. One of the pH sensors 632 is covered by a membrane layer 6312containing the enzyme urease. The urease hydrolyzes the urea to formcarbon dioxide and ammonia, in accordance with the following reaction:

(NH₂)₂CO+H₂O→2NH₃+CO₂

The resulting change in pH within the membrane 6312 is indicated by thechange in potential (in millivolts) as a function of the concentrationof urea. The second pH sensor 634 serves as a baseline to remove theeffect of changing sample pH. The sensors 632,634 may be conventionalelectrodes or pH sensitive ISFETs (ion selective field effecttransistors), such as those described in Chapter 7 (“Semiconductor FieldEffect Devices by F. Winquist and B. Danielsson) of Biosensors—APractical Approach, edited by A. E. G. Cass, IRL Press, 1990.

The power, logic and communication (telemetry) components 632 of thesensor are much the same as those described above for the glucose sensorof Example 7, including the memory device and microprocessor. Includedwithin the memory device can be the patient's identification, prescribedtreatment, and urea monitoring results. The sensor will store theresults of each treatment and this treatment history would be availableon demand or as a monthly report. The memory device can be sealed into awall of the “T”-connector 636, or can be a plug that is inserted througha dedicated opening in a wall of the connector, in either case,positioned so that the sensor is in direct contact with the fluid.

EXAMPLE 10

Free Calcium Sensor #1

The concentration of free calcium (Ca⁺⁺) in the blood, i.e., calciumthat is not bound to proteins, can be used to detect and monitor anumber of bone diseases or calcium regulation disorders (thyroid orkidney diseases). The test for free calcium typically involves drawingblood from a vein, then running laboratory tests to measure the amountof free calcium. The accuracy of such tests is affected by the patient'sconsumption of food or ingestion of or from administration ofmedications containing calcium within several hours prior to drawing theblood. The tests, thus, can provide incorrect results if the patient hasnot been careful about what was eaten, or is unaware that a particularfood could effect calcium blood level. Long term periodic monitoringwill be helpful not only in avoiding the need to repeat tests when theresults were not as expected, but also to determine the rate of changein calcium blood level over extended periods of time to determinewhether the patient's condition was worsening or improving.

Fluorescence-based techniques can be used to measure calciumconcentrations, and such techniques have reportedly made measurement ofintracellular calcium “routine” [see, e.g., Czarnik, (1995) “DesperatelySeeking Sensors” Chemistry & Biology 2:423-428.] Calcium-sensitivefluorescent agents, such as fura-2 and indo-1 are not consumed, sensing,and, thus, can be reused if the sensor material has not beenirreversibly commingled with the test sample. These, thus, can be usedas in sensors for measuring calcium.

A memory device, such as any of those described here, can be constructedcan be implanted for in vivo monitoring of calcium concentration in theblood, or can be sealed into a vial or other container in which blood tobe analyzed is received for in vitro monitoring. An optical detector,similar to that described for Example 8, will be connected to the power,logic and communication electronics. Unlike the sensor of Example 8, alight source will be selected to emit light not at the same wavelengthas that detected by the detector, but rather at the wavelength necessaryfor excitation of the fluorosensor [fluorophore]. The detector is thenselected to detect light at the wavelengths at which the fluorosensorfluoresces.

The entire sensor structure can be encased in a protective shell formedof an optically transparent plastic or polymer, so that light can exitand enter the shell. The exterior of the shell is coated with a matrixwhich contains the fluorosensor. The matrix may be a latex (see, e.g.,Slomkowski, et al. (1995) “Two-dimensional Latex Assemblies and TheirPotential Application in Diagnostics, TRIP 3:297-304) or collagen orother suitable, preferably biocompatible support material. It may bedesirable to optimize the amount of light directed toward the detectorby positioning a ring or other structure with an interior reflectingsurface around the outside of the shell. The reflector must besufficiently open in structure to permit the analyte, Ca²⁺ to flowthrough it to come in contact with the fluorosensor, which event will berecorded by the photodetector and memory.

EXAMPLE 11 Sol-gel Encapsulated Biomolecules and Memories and Sol-gelSensors

Sol-gel biosensors are known [see, e.g., Dave, et al. (1995) “Sol-GelEncapsulation Methods for Biosensors”, Analytical Chemistry 67:1120] fora description of preparation of a suitable sol gels. Briefly, thesol-gel can be a porous silicate glass formed by hydrolysis of analkoxide precursor followed by condensation to yield a polymericoxo-bridged SiO₂ network. The corresponding alcohol molecules areliberated during the process. The initial hydrolysis andpolycondensation reactions in a localized region lead to formation ofcolloidal particles. The suspension containing these particles is the“sol”. As the interconnection between the particles increases, theviscosity of the sol starts to increase and leads to the formation of asolid gel. The nature of the polymeric gel that results can be regulatedto a certain extent by controlling the rates of the individual steps. Aslong as solvent remains in the structure, the gel continues to change,with the pores becoming smaller and the gel strength increasing as thestructure ages. Most of the water and methanol liberated as a result ofhydrolysis is retained in the gel. As the water evaporates, the gelmaterial shrinks to approximately one-eighth the original gel volume.Once the gel shows no further loss of pore liquid under ambientconditions, it can be considered stable.

For biomolecular encapsulation in sol-gel, a buffer is added to increasethe pH to biologically compatible values. The protein to be encapsulatedis added to the sol after partial hydrolysis of the precursor. As thestructure ages, the pores close around the protein molecules, trappingthem inside. This trapping also ensures that the protein molecules aredistributed uniformly throughout the structure and are isolated fromeach other. Reactions of the encapsulated proteins with the analyte arelimited to single molecules, optimizing efficiency and simplifyingcalculation of maximum sensitivity for a given amount of protein.

Free Calcium Sensor #2

Using a similar memory structure to that described in Example 10, acalcium sensor can be fabricated using calcium-dependent photoproteins,such as the Aequorin photoprotein.

The photoprotein, aequorin, isolated from the jellyfish, Aequorea, emitslight upon the addition of Ca²⁺ or other metal ion. The bioluminescencephotoprotein aequorin is isolated from a number of species of thejellyfish Aequorea. It is a 22 kilodalton [kD] molecular weight peptidecomplex [see, e.g., Shimomura et al. (1962) J. Cellular and Comp.Physiol. 59:233-238; Shimo mura et al. (1969) Biochemistry 8:3991-3997;Kohama et al. (1971) Biochemistry 10:4149-4152; and Shimomura et al.(1972) Biochemistry 11:1602-1608].

The aequorin photoprotein, which includes bound luciferin and boundoxygen that is released by Ca²⁺, does not require dissolved oxygen.Luminescence is triggered by calcium, which releases oxygen and thecoelentrazine substrate producing apoaqueorin. The aequorin system iswell known [see, e.g., Tsuji et al. (1986) “Site-specific mutagenesis ofthe calcium-binding photoprotein aequorin, ” Proc. Natl. Acad. Sci. USA83:8107-8111; Prasher et al. (1985) “Cloning and Expression of the cDNACoding for Aequorin, a Bioluminescent

Calcium-Binding Protein,” Biochemical and Biophysical ResearchCommunications 126:1259-1268; Prasher et al. (1986) Methods inEnzymology 133:288-297; Prasher, et al. (1987) “Sequence Comparisons ofcDNAs Encoding for Aequorin Isotypes,” Biochemistry 26:1326-1332;Charbonneau et al. (1985) “Amino Acid Sequence of the Calcium-DependentPhotoprotein Aequorin,” Biochemistry 24:6762-6771; Shimomura et al.(1981) “Resistivity to denaturation of the apoprotein of aequorin andreconstitution of the luminescent photoprotein from the partiallydenatured apoprotein,” Biochem. J. 199:825-828; Inouye et al. (1989) J.Biochem. 105:473-477; Inouye et al. (1986) “Expression of ApoaequorinComplementary DNA in Escherichia coli,” Biochemistry 25:8425-8429;Inouye et al. (1985) “Cloning and sequence analysis of cDNA for theluminescent protein aequorin,” Proc. Natl. Acad. Sci. USA 82:3154-3158;Prendergast, et al. (1978) “Chemical and Physical Properties of Aequorinand the Green Fluorescent Protein Isolated from Aequorea forskalea” J.Am. Chem. Soc. 17:3448-3453; European Patent Application 0 540 063 A1;European Patent Application 0 226 979 A2, European Patent Application 0245 093 A1 and European Patent Specification 0 245 093 B1; U.S. Pat. No.5,093,240; U.S. Pat. No. 5,360,728; U.S. Pat. No. 5,139,937; U.S. Pat.No. 5,422,266; U.S. Pat. No. 5,023,181; U.S. Pat. No. 5,162,227; and SEQID Nos. 5-13, which set forth DNA encoding the apoprotein; and a form,described in U.S. Pat. No. 5,162,227, European Patent Application 0 540063 A1 and Sealite Sciences Technical Report No. 3 (1994), iscommercially available from Sealite, Sciences, Bogart, GA as AQUALITE®].

The native protein contains oxygen and a heterocyclic compoundcoelenterazine, a luciferin, [see, below] noncovalently bound thereto.The protein contains three calcium binding sites. Upon addition of traceamounts Ca²⁺ [or other suitable metal ion, such as strontium] to thephotoprotein, it undergoes a conformational change the catalyzes theoxidation of the bound coelenterazine using the protein-bound oxygen.Energy from this oxidation is released as a flash of blue light,centered at 469 nm. Concentrations of calcium ions as low as 10⁻⁶ M aresufficient to trigger the oxidation reaction. Thus, exposure to Ca⁺²triggers the light reaction, so that production of light is a measure ofCa⁺² concentration. If the rate of the reaction is controlled, theaqueorin system is an ideal Ca⁺² sensor.

This problem has been solved by encapsulating the Aqueorin protein in asol gel to produce an optical sensor [Aylott et al. (1996) Abst.presented at the 3rd European Conference Optical Chemical Sensors andBiosensors, Zurich, Switzerland]. The photoprotein Aequorin wasencapsulated into sol gel matrix to provide to slow the interactionbetween the Aqueorin photoprotein and the Ca⁺² in the sample. Thispermits highly sensitive measurements of Ca⁺² concentration. As modifiedherein, the a photodetector and memory will be incorporated into thegel, thereby permitting the light events to be detected and monitored.

For implementation using the memories provided herein, the power, logicand control electronics are encased within a protective,chemical-impervious, light transmissive shell, then submerged in thestill-liquid sol, so that the shell is coated with the matrix material.Since the reaction produces its own light, a separate light source isnot required, and only a photodetector need be included in the device.The photodetector is selected to detect light at the wavelength emittedby the photoprotein. At this point, the photoprotein, in this case,aequorin, has already been added to the sol.

The resulting sensor may be implantable, or may be placed as a separateunit into a solution, or built into a solution-containing vessel, suchas a blood-drawing test tube.

Other sol-gel sensors with memories

Other types of photosensors similarly may be encapsulated in a sol-gelshell coating the electronic “capsule” for providing sensors fordetecting other chemicals. The chemicals which may be detected includedissolved oxygen (O₂) and carbon monoxide (CO₂), using hemoglobin (Hb)or myoglobin (Mb) encapsulated in the sol-gel, both of which reversiblybind O2 to generate direct optical emission at 436 nm. The response timeof the reaction is about one minute, and the sensor is stable for a fewdays. Dissolved nitric oxide (NO) can be optically detected usingmanganese myoglobin (MnMb). The presence of NO causes a sol-gel matrixcontaining the manganese myoglobin to emit light at 436 nm. This sensoris also stable for a few days. Dye-mediated detection of glucose andoxalate can be performed using the glucose oxidase and oxalate oxidase,respectively, with an enzyme co-factor (NADH), also encapsulated withinthe sol-gel. A glucose detector so constructed emits light at 510 nm andthe oxalate detector emits light at 590 nm. Both of these sensors arestable over several months.

EXAMPLE 12 Blood Donor Bag

In order to assure that all donated blood is properly identified andtested, blood bank personnel must complete a significant amount ofpaperwork and must perform careful checks and double-checks of thedonor's information and correct labeling of the bag(s) and vialscontaining the blood. Once properly identified, the vials are forwardedto a laboratory where it is separated and tested for a number of viraldiseases. This procedure could be greatly simplified, and perhaps evenautomated, except for the nurse who draws the blood, by affixing theabove-described sensors within the bags to record donor information,perform various tests on the blood, and automatically record the testresults for later reading.

An exemplary embodiment of the “smart” blood bag is provided in FIG. 64,with the sensor array 644 located at one side 646 of bag 642. A singlemicroprocessor 648 and memory 6410 are shown as this facilitates a onestep remote write and read step, however, a dedicated microprocessor andmemory can be provided for each individual sensor, each combinationhaving a unique address to permit distinction between the differentcombinations during write and read steps. The microprocessor 648, memory6410, battery 6412 and transceiver components 6414 are configured muchlike the corresponding components of Example 7, with connectionsprovided between the microprocessor 648 and each of the individualsensors. The temperature sensor of Example 7, or a similar sensor 6416,is also included, to record exposure of the sample to excessive heat,which might render the blood unusable and/or could lead to incorrecttest results, and to provide temperature compensation for those teststhat are temperature sensitive.

The infectious diseases for which the donor blood is typically testedinclude hepatitis B and C, HIV/AIDS, and human T-cell lymphotropic virus(HTLV). A sensor will be provided for each test performed, and multipletests may be performed for a single disease in some cases.

As is known, the test for identification of the viral diseases ofconcern are conducted by immunoassay, i.e., measurement of thebioactivity (specific binding) between an antigen and an antibody. Forhepatitis B, the tests may be for Hepatitis B surface antigen(Anti-HBs), or Hepatitis B core antibody (Anti-HBc); for Hepatitis C,the test is for Hepatitis C virus antibody (Anti-HCV); for HIV/AIDS, thedifferent tests are HIV-1/HIV-2 antibody, HIV-1 (Western blot), andHIV-1 p24 antigen; and for HTLV, the HTLV-1 antibody.

Most bioanalytic sensors are based upon the redox immobilization ofenzymes. These proteins catalyze the conversion of the analyte betweentwo redox states that can subsequently be detected at the electrode.Redox enzymes include two large groups: the oxidase enzymes and thedehydrogenase enzymes. Both types of enzyme catalysis can be mediated bya variety of species that act as electron acceptors. The most commonmediators used in biosensing techniques are ferrocene compounds. Suchmediators can be incorporated into electropolymerized films.

For each of the above tests, a three electrode arrangement as in Example7 is provided, i.e., a working electrode, a reference electrode and acounter electrode. Also as in Example 7, a membrane is placed over aportion of the electrodes to permit the electrical detection of immunereactants. This membrane may be as described for Example 7, or may beone of the electroactive polymers described below. The specific antigenis conjugated with the enzyme, and both are retained within the membraneover the electrodes. Enzymes that may be used include, for example,horseradish peroxidase, alkaline phosphatase, glucose oxidase andβ-galactosidase. An appropriate ferrocene compound incorporated into athin film can be placed between the buffer layer over the electrodes andthe enzyme/antigen membrane.

The microprocessor is programmed to scan the voltage applied to theworking electrode between a maximum and a minimum level at a fixed scanrate. The scan rate varies from a few millivolts (mV) per second to afew hundred millivolts per second. This procedure is commonly used inbiosensors, and is known as cyclic voltammetry [see, e.g., Foulds, etal. “Immunoelectrodes”, in Biosensors—A Practical Approach, edited by A.E. G. Cass, IRL Press, 1990]. The current is then measured at theworking electrode. This current has two components: a capacitive ornon-Faradic component arising from redistribution of charged and polarspecies at the electrode surface, and a Faradaic component resultingfrom exchange of electrons between the electrode and redox activespecies at the electrode surface. If the potential is sufficientlyoxidizing or reducing and the rate of electron transfer between theelectrode and the redox species in solution is sufficiently fast, theFaradaic current is controlled by the rate of diffusion of theelectroactive species to the electrode.

As the voltage is swept upwards (more positive) the oxygen concentrationdrops, indicated by an increase in current. The current reaches amaximum value, then decays. Upon reversal of the voltage, the currentwill again increase as a result of re-oxidation. The Faradaic componentof the current must be extracted from the combined current. Thenon-Faradaic component of the current can be determined by the tangentto the initial slope during the first sweep. These procedures can beprogrammed into the microprocessor, so that a final value can be storedin memory, or the raw data can be stored for later processing once it isretrieved by the remote reader.

Referring again to FIG. 64, once the bag is filled with blood, smallcapillary tubes 6418 distribute the blood to the different test sites.The capillary tubes are configured for one-way flow so that the smallamounts of blood separated out cannot leak back into the main part ofthe bag. After waiting the appropriate time for the tests beingconducted, the user scans the bag to verify donor identity and to obtainthe stored test results.

As is known, other types of testing may be used to produce electricalsignals which can generate data for storage indicative of the testresults of each of the immunoassays. Alternative sensors types includethermometric sensors [which may include the use of sequentially actingenzyme for amplification of the total enthalpy change], and opticalsensors, e.g., a Western Blot test, as further described below.

The Western Blot test uses a film strip upon which the proteins, such asthose from human immunodeficiency virus [HIV] are laid out vertically inorder of molecular size, from largest to smallest, on a strip of film orpaper. Such a strip 652 is embedded in the test area of the blood bagwith open space over the strip for exposing the strip to the blood, asillustrated in FIG. 65. The appropriate enzyme is contained within arelease control system which is activated by a signal generated by thedevice's microprocessor within the device's electronics 654. Theelectroresponsive hydrogel described in U.S. Pat. No. 5,152,758 toKaetsu, et al. is one such control system. The electroactive polymersdescribed by J. N. Barisci, et al in “Conducting Polymer Sensors”, TRIP,Vol.4, No. 9, September 1996, pp. 307-311, may be utilized in a similarmanner. These materials are polymers into which physiologically activesubstances, including enzymes, can be incorporated during or aftercopolymerization, and which will release the physiologically activesubstance upon application of a voltage. In this case, theenzyme-containing polymer 656 is coated onto an electrode 658 which ispositioned adjacent the film 652. Release of the enzyme is timed by themicroprocessor to occur after a sufficient delay to permit binding ofany HIV antibodies and with the antigens. A second electrode 6510 coatedwith an electroactive polymer 6512 containing appropriate chemical dyeis used to release the chemical dye for marking the bound antibodies andantigens.

The strip film 652 can have a reflective backing or can be transparentor opaque. For a reflective backing, a photodetector 6514 positionedover the strip will measure the amount of light reflected. For atransparent strip or opaque, the photodetector is positioned behind thestrip. A second, reference photodetector 6516 is provided to provide abaseline measurement of the light intensity, which can be ambient light,light from an external light source, or light from an LED included inthe sensor device. The areas of the strip that are darkened 6520 by thechemical dye will absorb more light, reducing the total reflectedintensity. An electrical signal representative of the detected level oflight is then written into the device's memory for comparison to athreshold indicative of HIV infected blood, the threshold beingdetermined statistically based upon the minimum surface area of thestrip that will be darkened in a positive test.

EXAMPLE 13

Glucose Sensor #2

The power, logic and communication electronics of Example 7 are againused in this embodiment of a glucose sensor, which is illustrated inFIG. 66. The electrodes 664,666,668, however, are formed on the surfaceof the shell 662 which surround the electronics, providing a morecompact, implantable sensor. The shell is coated with an electroactivepolymer such a polypyrrole, a polythiophene, or a polyaniline [see,Barisci et al. (1996) “Conducting Polymer Sensors”, TRIP 4:307-311 for adescription of such polymers]. The use of electroactive polymers permitsthe direct incorporation of the catalyzing enzyme into the polymer,rather than requiring the physical formation of a well or cavity inwhich an enzyme-containing membrane is to be retained.

For a glucose sensor, a polypyrrole (PPy) coating is formed on thesurface of shell 662, through which conductive connectors 6610 extendfor connection to the electrodes. The electrodes 664,666,668 are definedby selective laser ablation of the coating to remove the materialbetween the electrodes. Alternatively, photolithographic techniques maybe used. The GOx is then adsorbed into the electrodes. It may also bedesirable to incorporate a ferrocene compound as a mediator into theelectrodes. Operation of the PPy electrode-based sensor will be similarto the procedure for the sensor described in Example 7.

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

What is claimed:
 1. A library of molecules or biological particlescomprising: a plurality of unique molecules or biological particles,wherein each molecule or biological particle is linked to andsynthesized on one of a plurality of discrete solid supports, whereineach solid support comprises a combination of: a matrix comprising amaterial treated for linking molecules or biological particles and aninformation surface for imprinting or engraving; and anoptically-readable symbol imprinted or engraved on the informationsurface, the symbol comprising optically-encoded data including a uniqueidentifier and an orientation indicator for determining orientation ofthe matrix with respect to an optical reader; wherein the uniqueidentifier corresponds to the molecule or biological particle that islinked to and synthesized on the material for linking so that eachmolecule or biological particle of the library is uniquely labeled. 2.The library of claim 1, wherein the matrix comprises a porousnon-collapsible vessel formed from an inert material and the materialfor linking comprises a plurality of particles disposed within thevessel.
 3. The library of claim 2, wherein the particles are derivatizedfor linking the molecules or biological particles.
 4. The library ofclaim 2, wherein the vessel comprises a frame formed from an inertmaterial which supports an inert mesh and a cap inserted into the frame.5. The library of claim 4, wherein an exterior surface of the capcomprises the information surface.
 6. The library of claim 2, whereinthe vessel has a volume of about 30 mm³ or less.
 7. The library of claim1, wherein the information surface is formed from a material that isdifferent from the material for linking.
 8. The library of claim 1,wherein the information surface is formed from a material selected fromthe group consisting of polypropylene, glass, polytetrafluoroethylene,polyethylene, polystyrene, polyester, ceramic or composites thereof. 9.The library of claim 1, wherein the material for linking is aradiation-grafted polymer that is derivatized for linking the moleculesor biological particles.
 10. The library of claim 1, wherein theoptically-readable symbol is a two-dimensional bar code.
 11. The libraryof claim 1, wherein the optically-readable symbol comprises a pluralityof holes or pits formed in the information surface.
 12. The library ofclaim 1, wherein the molecules or biological particles are selected fromthe group consisting of monomers, nucleotides, amino acids, smallmolecules, antigens, antibodies, ligands, proteins, nucleic acids, phageand viral particles, and cells.
 13. The library of claim 1 that is acombinatorial library.
 14. A library of molecules or biologicalparticles comprising: a plurality of unique molecules or biologicalparticles, wherein each molecule or biological particle is linked to andsynthesized on one of a plurality of discrete solid support devices,each solid support device comprising the combination of: a vesselcomprising a frame formed from an inert material and an inert meshsupported by the frame, the mesh adapted to permit reagents to passtherethrough; wherein the vessel has an information surface; a pluralityof particles contained within the vessel, wherein the particles aretreated for linking molecules or biological particles; and anoptically-readable symbol imprinted or engraved on the informationsurface, the symbol comprising optically-encoded data including a uniqueidentifier and an orientation indicator for determining orientation ofthe symbol with respect to an optical reader; wherein the uniqueidentifier corresponds to the molecule or biological particle that islinked to and synthesized on the particles contained within the vesselso that each molecule or biological particle of the library is uniquelylabeled.
 15. The library of claim 14, wherein the frame is adapted forreceiving a cap for sealing the vessel.
 16. The library of claim 15,wherein an outer surface of the cap comprises the information surface.17. The library of claim 14, wherein the vessel has a volume of about 30mm³ or less.
 18. The library of claim 14, wherein the optically-readablesymbol is a two-dimensional bar code.
 19. The library of claim 14,wherein the molecules or biological particles are selected from thegroup consisting of monomers, nucleotides, amino acids, small molecules,antigens, antibodies, ligands, proteins, nucleic acids, phage and viralparticles, and cells.
 20. The library of claim 14 that is acombinatorial library.